Methods for generating antibiotic resistant microbes and novel antibiotics

ABSTRACT

The invention provides methods for generating antibiotic resistant bacteria comprising blocking mismatch repair in a bacterium to make hypermutable bacteria, contacting the bacteria with at least one antibiotic, selecting bacteria that are resistant to the antibiotic, and culturing the antibiotic resistant bacteria. The invention also provides methods of determining the genes responsible for antibiotic resistance.

FIELD OF THE INVENTION

[0001] This invention relates to the field of antimicrobial treatmentsand gene targets for the discovery of antimicrobial agents. Inparticular, it relates to the discovery of genes essential for growthand virulence of bacteria.

[0002] Despite the development of new classes of antimicrobial agentsover the past decade (reviewed in http://vet.purdue.edu/bms), microbialinfections remain a serious health problem. While antibiotics treatmenthas been effective in controlling infectious diseases, an increase inthe number of antibiotic-resistant (AR) microbes have emerged and arenow posing a major therapeutic problem. In today's industrializedsocieties, infectious strains can be found that are resistant to allclasses of antimicrobial agents used in the clinic. Infections due toresistant strains include higher morbidity and mortality, longer patienthospitalization, and an increase in treatment costs (Murray (1994) NewEngl. J. Med. 330:1229-1230). In light of these findings, an unmet needexists for the development of new therapeutic agents that can work byinhibiting the ever-increasing number of novel antibiotic resistancemechanisms.

[0003] One approach for generating new therapies and/or therapeuticstrategies against AR microbes is to develop methods that can generate awide array of genomic alterations in a microbe's genome that can yieldmaximal number altered target genes that are capable of elicitingantibiotic resistance. Once an AR strain is developed, it can be usedfor rapid genome analysis to identify mutant gene(s) that are capable ofrendering a microbe resistant to an antibiotic for targetidentification. Such analysis can involve any of a variety of methodsused by those skilled in the art for identifying mutations and/ordifferential gene expression, including but not limited to differentialgene expression using microarrays, cDNA subtraction, differentialprotein analysis, complementation assays, single nucleotide polymorphosm(SNP) analysis or whole genome sequencing to identify altered loci.

[0004] A bottleneck to generating genetically diverse microbes is theinability to generate nonbiased genome-wide mutations. Many mutagenesismethods are available whereby the use of chemical and radiation exposurehas been successful in generating genomic mutations. A limitation ofthis approach is that these various methods are usually DNA sitespecific or are extremely toxic, therefore limiting the mutation spectraand the opportunity to identify a maximal number of genes, when mutated,that are able to confer resistance to an antibiotic. Recently, work doneby Nicolaides, et al. (Nicolaides et al. (1998) Mol. Cell. Biol.18:1635-1641; U.S. Pat. No. 6,146,894) has demonstrated the utility ofintroducing dominant negative mismatch repair mutants into cells toconfer global DNA hypermutability. These mutations are in the form ofpoint mutations or small insertion-deletions that are distributedequally throughout the genome. The ability to manipulate the mismatchrepair (MMR) process of a target host organism can lead to an increasein the mutability of the target host genome, leading to the generationof innovative cell subtypes with varying phenotypes from the originalwild-type cells. Variants can be placed under a specified, desiredselective process the result of which is the capacity to select for anovel organism that expresses an altered biological molecule(s) and hasa new phenotype. The concept of creating and introducing dominantnegative allele of a gene, including the MMR alleles, in bacterial cellshas been documented to result in genetically altered prokaryoticmismatch repair genes (Aronshtam and Marinus (1996) Nucl. Acids Res.24:2498-2504; Wu and Marinus (1994) J. Bacteriol. 176:5393-400; Broshand Matson (1995) J. Bacteriol. 177:5612-5621). Furthermore, altered MMRactivity has been demonstrated when MMR genes from different speciesincluding yeast and mammalian cells are over-expressed (Fishel et al.(1993) Cell 7:1027-1038; Lipkin et al. (2000) Nat. Genet. 24:27-35). Theability to create hypermutable organisms by blocking MMR has greatcommercial value for the generation of AR bacterial strains for drugscreening and target discovery.

[0005] There is an urgent need in the art to elucidate the mechanisms ofantimicrobial resistance, and to identify novel antimicrobial agents.

SUMMARY

[0006] The invention provides new uses of MMR deficiency in bacteria toidentify antibiotic resistance (AR) genes and pathways that can lead tothe generation of novel therapeutic strategies for alternative clinicalstrategies.

[0007] Antibiotic resistant (AR) microbes express a number of genes thatare essential for growth of the organism in an infection, and serve asuseful reagents for target discovery and/or screening lines for thediscovery of novel antimicrobial agents. This invention provides anapproach to the identification of genes that confer anti-microbialresistance, and the use of those genes, and bacterial strains expressingmutant forms of genes, in the identification, characterization, andevaluation of targets for therapeutic development. In addition, thisapplication teaches of the use of employing structural information ofthe gene, gene product and mutant strains in screening for antimicrobialagents active against the genes and their corresponding products andpathways. Positive compounds can then be used as final products orprecursors to be further developed into antibacterial agents. Thisinvention also provides methods of treating microbial infections inmammals by administering an antimicrobial agent active against such anidentified target gene or product, and the pharmaceutical compositionseffective for such treatment.

[0008] To identify genes capable of rendering bacteriaantibiotic-resistant, the invention provides methods of decreasing MMRactivity of a microbial host to produce AR strains. Using this process,commercially viable microbes that are resistant to a wide range ofantibiotics can be directly selected for the resistance to ananti-microbial agent of interest. AR microbes may be geneticallyscreened to identify novel therapeutic targets for drug development.Once a bacterium with a specified resistance is isolated, the MMRactivity may be restored by several methods well known to those skilledin the art as a means to gentically “fix” the new mutations in the hostgenome, thereby making the AR microbe suitable for comparative molecularanalysis to the wild-type strain as well as for drug screening toidentify novel antimicrobial compounds. For example, if MMR is decreasedby the use of a dominant-negative allele or antisense vector directed toan internal MMR gene, the endogenous repair activity can be restored ifthe gene is expressed by an inducible promoter system, including but notlimited to promoters such as: TAC-LACI, tryp (Brosius et al. (1984) Gene27:161-172), araBAD (Guzman et al. (1995) J. Bact. 177:4121-4130) pLex(La Vallie et al. (1992) Bio. Technology 11:187-193), pRSET (Schoepfer,R. (1993) Gene 124:83-85), pT7 (Studier (1991) J. Mol. Biol.219(l):37-44) etc., by removing the inducer and, therefore, reducing thethe promoter activity. In the case that the expression vector employs aCre-lox system, MMR can be restored by disrupting the cDNA gene insertfrom the host cell harboring the expression vector (Hasan, N. et al.(1994) Gene 2:51-56). Yet other methods include homologous knockout ofthe expression vector that can turn off the actively expressed gene usedto inhibit MMR activity. In addition to the recombinant methods outlinedabove that have the capacity to eliminate the MMR activity from themicrobe, it has been demonstrated that many chemicals have the abilityto “cure” microbial cells of plasmids. For example, chemical treatmentof cells with drugs including bleomycin (Attfield et al. (1985)Antimicrob. Agents Chemother. 27:985-988) or novobiocin, coumercycin,and quinolones (Fu et al. (1988) Chemotherapy 34:415-418) have beenshown to result in microbial cells that lack endogenous plasmid asevidenced by Southern analysis of cured cells as well as sensitivity tothe appropriate antibiotic (Attfield et al. (1985) Antimicrob. AgentsChemother. 27(6):985-988, Fu et al. (1988) Chem. Abstracts34(5):415-418; BiWang et al. (1999) J. of Fujian Agricultural University28(1):43-46; Brosius, J. (1988) Biotechnology 10:205-225). Whether byuse of recombinant means or treatment of cells with chemicals, removalof the MMR-expression plasmid results in the reestablishment of agenetically stable microbial cell line. Therefore, the restoration ofMMR allows host bacteria to function normally to repair DNA. The newlygenerated mutant bacterial strain that exhibits a novel anti-microbialresistance is now suitable for gene/protein discovery to identify newbiomolecules that are involved in generating resistance as well as amodel system to screen for novel anti-microbial agents targeted againstcertain antibiotic resistant strains.

[0009] In certain embodiments, the invention provides methods forgenerating antibiotic resistant bacteria comprising the steps of:

[0010] blocking mismatch repair in the bacterium whereby the bacteriumbecomes hypermutable;

[0011] contacting the bacterium with at least one antibiotic determiningwhether the bacterium is resistant to the antibiotic, thereby generatingantibiotic resistant bacteria.

[0012] In the methods of the invention, mismatch repair may be blockedin some embodiments by introducing a polynucleotide encoding a wild-typeallele of a mismatch repair gene into a cell, whereby the wild-typeallele inactivates the endogenous MMR activity by binding to andinterfering with the resident activity. The cell becomes hypermutable asa result of the introduction of the gene.

[0013] In other embodiments of the invention, a polynucleotide encodinga dominant negative allele of a mismatch repair gene is introduced intoa cell, where the dominant negative gene is derived from a mismatchrepair gene from a different organism. The cell becomes hypermutable asa result of the introduction of the gene. In particular embodiments ofthis method, MMR activity is inhibited for ten rounds of cell divisionand then the MMR activity is restored therefore restoring the geneticstability. An example of a dominant negative MMR gene is the PMS2-134gene.

[0014] In other embodiments of the invention, a polynucleotide encodingan allele of a mismatch repair gene is introduced into a bacterial cell,where the mismatch repair gene is derived from a wild-type or alteredmammalian, yeast, fungal, amphibian, insect, plant or bacterial mismatchrepair gene. The cell becomes hypermutable as a result of theintroduction of the gene.

[0015] In another embodiment, mismatch repair may be blocked byintroducing an antisense nucleic acid molecule into the bacteriumwherein the antisense nucleic acid molecule specifically binds to amismatch repair gene and inhibits mismatch repair in the bacterium.

[0016] In other embodiments of the invention, methods are provided forgenerating a genetic alteration of a bacterial host genome to producevariant strains expressing new output traits. Transgenic bacteriumcomprising a polynucleotide encoding a wild-type allele of a mismatchrepair gene is grown. The bacteria are comprised of a set of alteredgenes for a desired biological phenotype.

[0017] In other embodiments of the invention, methods are provided forgenerating a genetic alteration of a bacterial host genome to producevariant strains that are resistant to antimicrobial agents. Bacteriawith decreased mismatch repair are grown. The bacteria are comprised ofa set of altered genes for a desired antibiotic-resistance phenotype.

[0018] In further embodiments of the invention, methods are provided forcreating a hypermutable bacterium using a wild-type MMR allele forantibiotic-resistance selection, and restoring genomic stability of aselected host by inactivating or decreasing the expression of thewild-type MMR allele.

[0019] In another embodiment of the invention, a method is provided forcreating a hypermutable bacteria using a dominant negative MMR allelefor antibiotic-resistance selection, and restoring genomic stability ofa selected host by inactivating or decreasing the expression of thedominant negative MMR gene allele.

[0020] In another embodiment of the invention, a method is provided forcreating a hypermutable bacteria expressing an antisense gene to a MMRgene for antibiotic-resistance selection, and restoring genomicstability of a selected host by inactivating or decreasing theexpression of the dominant negative MMR gene allele.

[0021] In another embodiment of the invention, a method is provided forcreating a hypermutable bacteria using chemical inhibitors of MMR forantibiotic-resistance selection, and restoring genomic stability of aselected host by removing the chemical inhibitor by introducing adominant negative allele of a mismatch repair gene into the bacterium.The dominant negative allele may be, for example, a PMS2-134 gene.

[0022] In another embodiment, mismatch repair may be blocked by exposingthe bacterium a to a compound that inhibits mismatch repair wherebycells are grown in the presence of the compound and undergo multiplerounds of cell divison in the absence of MMR, yielding sibs that aregenetically diverse. Sibs are then selected for antibiotic resistance.AR strains are removed from chemical inhibitor and the endogenous MMRactivity is restored leaving genetically stable strains that are nowsuitable for gene discovery and/or therapeutic agent development. Forexample, the compound that blocks mismatch repair may be an anthracenederivative, including, but not limited 1,2-dimethylanthracene,9,10-dimethyl anthracene, 7,8-dimethylanthracene,9,10-diphenylanthracene, 9,10-dihydroxymethylanthracene,9-hydroxymethyl-10-methylanthracene, dimethylanthracene-1,2-diol,9-hydroxymethyl-10-methylanthracene-1,2-diol,9-hydroxymethyl-10-methylanthracene-3,4-diol, 9, 10-di-m-tolyanthracene.In other embodiments, the compound that blocks MMR activity is an ATPanalog. In other embodiments, the compound that blocks MMR activity is anuclease inhibitor. In other embodiments, the compound that blocks MMRactivity is a DNA polymerase inhibitor.

[0023] The methods of the invention may further comprise exposing thebacteria to chemical mutagens. While it has been documented that MMRdeficiency can lead to as much as a 1000-fold increase in the endogenousDNA mutation rate of a host, there is no assurance that MMR deficiencyalone will be sufficient to alter every gene within the DNA of the hostbacterium to create altered biochemicals with new activity(s).Therefore, the use of chemical agents and their respective analoguessuch as methane sulfonate, dimethyl sulfonate, O-6-methyl benzadine,ethylnitrosourea (ENU), ethidium bromide, ethyl methanesulfonate (EMS),N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), methylnitrosourea (MNU),Tamoxifen, 8-hydroxyguanine, as well as others listed but not limited toin publications by: Khromov-Borisov et al (1999) Mutat. Res. 430:55-74;Ohe et al. (1999) Mutat. Res. 429:189-199; Hour et al. (1999) Food Chem.Toxicol. 37:569-579; Hrelia et al. (1999) Chem. Biol. Interact.118:99-111; Garganta et al. (1999) Environ. Mol. Mutagen. 33:75-85;Ukawa-Ishikawa et al (1998) Mutat. Res. 412:99-107;www.ehs.utah.edu/ohh/mutagens, etc. can be used in the methods of theinvention to further enhance the spectrum of mutations and increase thelikelihood of obtaining alterations in one or more genes that can inturn generate host bacteria with a complex antibiotic resistantphenotype (Fu et al. (1988) Chemotherapy 34(5):415-418; Lee et al.(1994) Mutagenesis 9:401-405; Vidal et al. (1995) Carcinogenesis16:817-821). Prior art teaches us that mismatch repair deficiency leadsto hosts with an increased resistance to toxicity by chemicals with DNAdamaging activity. This feature allows for the creation of additionalgenetically diverse hosts when MMR defective bacteria are exposed tosuch agents, which would be otherwise impossible due to the toxiceffects of such chemical mutagens (Colella et al. (1999) Br. J. Cancer80:338-343; Moreland et al. (1999) Cancer Res. 59:2102-2106; Humbert etal. (1999) Carcinogenesis 20:205-214; Glaab et al. (1998) Mutat. Res.398:197-207). Moreover, prior art teaches us that MMR is responsible forrepairing chemical-induced DNA adducts, so therefore blocking thisprocess could theoretically increase the number, types, mutation rateand genomic alterations of a bacterial host [Rasmussen et al. (1996)Carcinogenesis 17:2085-2088; Sledziewska-Gojska et al. (1997) Mutat.Res. 383:31-37; Janion et al. (1989) Mutat. Res. 210:15-22). In additionto the chemicals listed above, other types of DNA mutagens includeionizing radiation and UV-irradiation, which are known to cause DNAmutagenesis in bacteria can also be used to potentially enhance thisprocess. These agents, which are extremely toxic to host cells and,therefore, result in a decrease in the actual pool size of alteredbacterial cells, are more tolerated in MMR defective hosts and in turnallow for a enriched spectrum and degree of genomic mutation ( Drummondet al. (1996) J. Biol. Chem. 271:9645-19648). such as, but not limitedto methane sulfonate, dimethyl sulfonate, O-6-methyl benzadine,ethylnitrosourea, ethidium bromide, ethyl methanesulfonate,N-methyl-N′-nitro-N-nitrosoguanidine, methylnitrosourea, Tamoxifen, and8-hydroxyguanine.

[0024] The methods of the invention may be used to generate AR bacteriawhich are resistant to such antibiotic compounds as, for example,quinilones, aminoglycosides, magainins, defensins, tetracyclines,beta-lactams, macrolides, lincosamide, sulfonamides, chloramphenicols,nitrofurantoins, and isoniazids.

[0025] In the methods of the invention, the step of determining whetherthe bacterium is resistant to an antibiotic may comprise analyzing thebacterium for multiantiboitic resistance.

[0026] Further, the methods of the invention may comprise makingantibiotic resistant bacteria genetically stable, such as by removingthe MMR inhibitory molecule, for example.

[0027] In the methods of the invention, the genome of the antibioticresistant bacterium and the genome of a wild-type strain of thebacterium may be compared by sequence analysis of the entire genomes, orcompared by microarray analysis, for example.

[0028] In another embodiment, the genome of said antibiotic resistantbacterium and the genome of said wild-type strain of said bacterium arecompared by:

[0029] introducing gene fragments from the antibiotic resistantbacterium into the wild-type bacterium, thereby producing mutantbacteria;

[0030] selecting a mutant bacterium with antibiotic resistance; andsequencing the gene fragment from the mutant bacterium with antibioticresistance, thereby identifying the antibiotic resistant gene.

[0031] The invention also provides methods of using microbial strainsthat are naturally defective for MMR due to defects in genes encodingfor MMR proteins. Strains in which muts, mutL, muth, or mutY genes aredefective have been reported to be defective in MMR activity (Modrich(1994) Science 266:1959-1960). The methods of the invention may employbacterial strains with mutant endogenous MMR genes for selecting forvariants that are AR. Once an AR variant strain is identified, thegenetic stability of the microbe can be restored by expressing afunctional gene that can complement the defective MMR gene activity.

[0032] Mutant strains can be used for gene identification by isolatingDNA fragments derived from the MMR defective antibiotic-resistantstrains. These bacteria contain DNA fragments with altered sequencesthat can be introduced into wild-type counterparts (antibioticsusceptible) and screened for fragments that confer antibioticresistance. Conversely, DNA fragments derived from the wild-typebacteria can be introduced into mutant bacterial strains to screen forgenes effective via loss-of-function mutated genes. The fact that aclone is complemented suggests the introduced fragment contains a geneencoding for an antibiotic-resistant gene product. Other methods canalso be used to identify AR genes including but not limited tomicroarray analysis of gene expression, differential expression and/ordifferential protein analysis know by those skilled in the art.

[0033] The microbial strains described herein have either been generatedand characterized in a manner which essentially provides a process bywhich the manipulation of MMR can confer AR against a wide range ofanti-microbial compounds and that these strains are now useful fortarget discovery and/or therapeutic agent discovery as screening lines.

[0034] In other embodiments of the invention, methods of producing astable bacterium expressing a new phenotype is provided. Turning off theexpression of the MMR-wild-type alleles, MMR-dominant negative alleles,or MMR-antisense alleles, results in genetically stable bacteriaexpressing a new output trait(s).

[0035] The invention also provides antibiotic resistant strains ofbacteria produced by the methods of the invention.

[0036] These and other aspects of the invention provide the art withmethods that can generate enhanced mutability in bacteria as well asproviding prokaryotic organisms harboring potentially useful mutationsto generate novel output traits for commercial applications, and are setforth in greater detail below.

BRIEF DESCRIPTION OF THE FIGURE

[0037]FIG. 1 shows growth of tetracyclin-resistant mutant bacteriacarrying a dominant negative allele of PMS2 in the pT7Ea plasmid(134/V5), tetracyclin-resistant mutant bacteria carrying a the PMSR3gene in the pT7Ea plasmid (R3), and wild-type bacteria carrying theempty pT7Ea plasmid (T7), on medium containing tetracyclin at 0, 4 and 6hours after tetracycline addition.

DETAILED DESCRIPTION OF THE INVENTION

[0038] The inventors present a method for developing hypermutablebacteria by altering the activity of endogenous mismatch repair (MMR)activity of hosts to generate antibiotic resistant (AR) microbes fortarget discovery and the development of novel anti-microbial agent byscreening for new compounds. Wild-type and some dominant negativealleles of mismatch repair genes, when introduced and expressed inbacteria, increase the rate of spontaneous mutations by reducing theeffectiveness of the endogenous MMR-mediated DNA repair activity,thereby rendering the bacteria highly susceptible to genetic alterationsdue to hypermutability. Hypermutable bacteria can then be utilized toscreen for novel mutations in a gene or a set of genes that producevariant siblings exhibiting new output traits not found in the wild-typecells such as antibiotic resistance.

[0039] The process of mismatch repair, also called mismatchproofreading, is an evolutionarily highly conserved process that iscarried out by protein complexes described in cells as disparate asprokaryotic cells such as bacteria to more complex mammalian cells(Modrich (1994) Science 266:1959-1960; Strand et al. (1993) Nature365:274-276; Su et al. (1988) J. Biol. Chem. 263:6829-6835;Aronshtam.and Marinus (1996) Nucl. Acids Res. 24:2498-2504; Wu andMarinus (1994) J. Bacteriol. 176:5393-400). A mismatch repair gene is agene that encodes one of the proteins of such a mismatch repair complex.Although not wanting to be bound by any particular theory of mechanismof action, a mismatch repair complex is believed to detect distortionsof the DNA helix resulting from non-complementary pairing of nucleotidebases. The non-complementary base on the newer DNA strand is excised,and the excised base is replaced with the appropriate base that iscomplementary to the older DNA strand. In this way, cells eliminate manymutations that occur as a result of mistakes in DNA replication,resulting in genetic stability of the sibling cells derived from theparental cell.

[0040] Some wild-type MMR gene alleles as well as dominant negativealleles cause a mismatch repair defective phenotype even in the presenceof a wild-type MMR gene allele in the same cell. An example of adominant negative allele of a MMR gene is the human gene hPMS2-134,which carries a truncation mutation at codon 134 (Nicolaides et al.(1998) Mol. Cell. Biol. 18:1635-1641). The mutation causes the productof this gene to abnormally terminate at the position of the 134th aminoacid, resulting in a shortened polypeptide containing the N-terminal 133amino acids. Such a mutation causes an increase in the rate ofmutations, which accumulate in cells after DNA replication. Expressionof a dominant negative allele of a mismatch repair gene results inimpairment of mismatch repair activity, even in the presence of thewild-type allele. Any mismatch repair allele, which produces sucheffect, can be used in this invention. In addition, the use ofover-expressed wild-type MMR gene alleles from human, mouse, plants, andyeast in bacteria has been shown to cause a dominant negative effect onthe bacterial hosts MMR activity (Fishel et al. (1993) Cell 7:1027-1038;Aronshtam and Marinus (1996) Nucl. Acids Res. 24:2498-2504; Wu andMarinus (1994) J. Bacteriol. 176:5393-400; Lipkin et al. (2000) Nat.Genet. 24:27-35).

[0041] Dominant negative alleles of a mismatch repair gene can beobtained from the cells of humans, animals, yeast, bacteria, plants orother organisms. Screening cells for defective mismatch repair activitycan identify such alleles. Mismatch repair genes may be mutant orwild-type. Bacterial host MMR may be mutated or not. The term bacteriaused in this application include any organism from the prokaryotickingdom. These organisms include genera such as but not limited toAgrobacterium, Anaerobacter, Aquabacterium, Azorhizobium, Bacillus,Bradyrhizobium, Cryobacterium, Escherichia, Enterococcus,Heliobacterium, Klebsiella, Lactobacillus, Methanococcus,Methanothermobacter, Micrococcus, Mycobacterium, Oceanomonas,Pseudomonas, Rhizobium, Staphylococcus, Streptococcus, Streptomyces,Thermusaquaticus, Thermaerobacter, Thermobacillus, etc. Otherprocaryotes that can be used for this application are listed at(www.bacterio.cict.fr/validgenericnames). Bacteria exposed to chemicalmutagens or radiation exposure can be screened for defective mismatchrepair. Genomic DNA, cDNA, or mRNA from any cell encoding a mismatchrepair protein can be analyzed for variations from the wild-typesequence. Dominant negative alleles of a mismatch repair gene can alsobe created artificially, for example, by producing variants of thehPMS2-134 allele or other mismatch repair genes (Nicolaides et al.(1998) Mol. Cell. Biol. 18:1635-1641). Various techniques ofsite-directed mutagenesis can be used. The suitability of such alleles,whether natural or artificial, for use in generating hypermutablebacteria can be evaluated by testing the mismatch repair activity (usingmethods described in Nicolaides et al. (1998) Mol. Cell. Biol.18:1635-1641) caused by the allele in the presence of one or morewild-type alleles, to determine if it is a dominant negative allele.

[0042] A bacterium that over-expresses a wild-type mismatch repairallele or a dominant negative allele of a mismatch repair gene willbecome hypermutable. This means that the spontaneous mutation rate ofsuch bacteria is elevated compared to bacteria without such alleles. Thedegree of elevation of the spontaneous mutation rate can be at least2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold,or 1000-fold that of the normal bacteria as measured as a function ofbacterial doubling/minute.

[0043] According to one aspect of the invention, a polynucleotideencoding either a wild-type or a dominant negative form of a mismatchrepair protein is introduced into bacteria. The gene can be any dominantnegative allele encoding a protein which is part of a mismatch repaircomplex, for example, muts, mutL, mutH, or mutYhomologs of thebacterial, yeast, plant or mammalian genes (Modrich (1994) Science266:1959-1960; Prolla et al. (1994) Science 264:1091-1093). The dominantnegative allele can be naturally occurring or made in the laboratory.The polynucleotide can be in the form of genomic DNA, cDNA, RNA, or achemically synthesized polynucleotide or polypeptide. The molecule canbe introduced into the cell by transfection or other methods welldescribed in the literature.

[0044] Transfection is any process whereby a polynucleotide orpolypeptide is introduced into a cell. The process of transfection canbe carried out in a bacterial culture using a suspension culture. Thebacteria can be any type classified under the prokaryotes.

[0045] In general, transfection will be carried out using a suspensionof cells but other methods can also be employed as long as a sufficientfraction of the treated cells incorporate the polynucleotide orpolypeptide so as to allow transfected cells to be grown and utilized.The protein product of the polynucleotide may be transiently or stablyexpressed in the cell. Techniques for transfection are well known tothose skilled in the art. Available techniques to introduce apolynucleotide or polypeptide into a prokaryote include but are notlimited to electroporation, transduction, cell fusion, the use ofchemically competent cells (e.g., calcium chloride), and packaging ofthe polynucleotide together with lipid for fusion with the cells ofinterest. Once a cell has been transformed with the inhibitory mismatchrepair gene or protein, the cell can be propagated and manipulated ineither liquid culture or on a solid agar matrix, such as a petri dish.If the transfected cell is stable, the gene will be retained andexpressed at a consistent level when the promoter is constitutivelyactive, or when in the presence of appropriate inducer molecules whenthe promoter is inducible, for many cell generations, and a stable,hypermutable bacterial strain results.

[0046] An isolated bacterial cell is a clone obtained from a pool of abacterial culture by chemically selecting out strains using antibioticselection of an expression vector. If the bacterial cell is derived froma single cell, it is defined as a clone.

[0047] Bacterial cultures may be screened for antibiotic resistanceagainst a wide array of antibiotic compounds. For example, but not byway of limitation, bacteria produced by the methods of the invention maybe screened for resistance to quinilones, aminoglycosides, magainins,defensins, tetracyclines, beta-lactams, macrolides, lincosamide,sulfonamides, chloramphenicols, nitrofurantoins, and isoniazids. Theantibiotics may be incorporated into solid or liquid growth medium, forexample.

[0048] A polynucleotide encoding an inhibitory form of a mismatch repairprotein can be introduced into the genome of a bacterium or propagatedon an extra-chromosomal plasmid. Selection of clones harboring themismatch repair gene expression vector can be accomplished by additionof any of several different antibiotics, including but not limited toampicillin, kanamycin, chloramphenicol, zeocin, and tetracycline. Themicrobe can be any species for which suitable techniques are availableto produce transgenic microorganisms, such as but not limited to generaincluding Bacillus, Pseudomonas, Staphylococcus, Escherichia and others.Any method for making transgenic bacteria known in the art can be used.According to one process of producing a transgenic microorganism, thepolynucleotide is transfected into the microbe by one of the methodswell known to those in the art. Next, the microbial culture is grownunder conditions that select for cells in which the polynucleotideencoding the mismatch repair gene is either incorporated into the hostgenome as a stable entity or propagated on a self-replicatingextra-chromosomal plasmid, and the protein encoded by the polynucleotidefragment transcribed and subsequently translated into a functionalprotein within the cell. Once a transgenic microbe is engineered toharbor the expression construct, it is then propagated to generate andsustain a culture of transgenic microbes indefinitely.

[0049] Once a stable, transgenic microorganism has been engineered toexpress a functional MMR protein, the microbe can be exploited to createnovel mutations in one or more target gene(s) of interest harboredwithin the same microorganism. A gene of interest can be any genenaturally possessed by the bacterium or one introduced into thebacterial host by standard recombinant DNA techniques. The targetgene(s) may be known prior to the selection or unknown. One advantage ofemploying such transgenic microbes to induce mutations in resident orextra-chromosomal genes within the microbe is that it is unnecessary toexpose the microorganism to mutagenic insult, whether it be chemical orradiation in nature, to produce a series of random gene alterations inthe target gene(s). This is due to the highly efficient nature and thespectrum of naturally occurring mutations that result as a consequenceof the altered mismatch repair process. However, it is possible toincrease the spectrum and frequency of mutations by the concomitant useof either chemical and/or radiation together with MMR defective cells.The net effect of the combination treatment is the increase in alteredgene pool in the genetically altered microbe that result in an increasedalteration of an allele(s) that are useful for producing new outputtraits. Other benefits of using MMR-defective microbes that are taughtin this application are genetic screens for the DIRECT selection ofvariant sub-clones that exhibit new output traits with commerciallyimportant applications such as antibiotic resistance, which allows thebypassing of the tedious and time consuming gene identification,isolation and characterization stages.

[0050] Mutations can be detected by analyzing the recombinant microbefor alterations in the genotype and/or phenotype post-activation of thedecreased mismatch repair activity of the transgenic microorganism.Novel genes that produce altered phenotypes in MMR-defective microbialcells can be discerned by any variety of molecular techniques well knownto those in the art. For example, the microbial genome can be isolatedand a library of restriction fragments cloned into a plasmid vector. Thelibrary can be introduced into a “normal” cell and the cells exhibitingthe novel phenotype screened. Transformed cells are then screened forthe new phenotype (e.g., antibiotic resistance). A plasmid is isolatedfrom those normal, transformed cells that exhibit the novel phenotypeand the inserted gene(s) characterized by DNA sequence analysis.

[0051] Alternatively, differential messenger RNA screen can be employedutilizing driver and tester RNA (derived from wild-type and novel mutantrespectively) followed by cloning the differential transcripts andcharacterizing them by standard molecular biology methods well known tothose skilled in the art. Furthermore, if the mutant sought is onencoded by an extrachromosmal plasmid, then following co-expression ofthe dominant negative MMR gene and the gene of interest to be alteredand phenotypic selection, the plasmid is isolated from mutant clones andanalyzed by DNA sequence analysis by methods well known to those in theart.

[0052] In another embodiment, the screening of cells may be performed bymicroarray analysis. In microarray analysis, microchips containing allor a subset of all expressed bacterial genes may be screened using RNAmolecules derived from the wild-type or antibiotic resistant strainwhereby RNA derived from one strain is reverse transcribed usingFluoroLink Cy3 and the other RNA sample is reverse transcribe-labelledusing Cy5 dUTP. Labelled cDNAs from each organism are used to probe themicrochip whereby unique message from one source will generate adistinct signal while message expressed from both sources will generatea common fluorescence. Alternatively, microchips containingolignucleotide derived from the wild-type strain can be used tohybridize genomic fragments from the antibiotic resistant strain toidentify fragments containing a mutated gene by loss of hybridization.

[0053] Phenotypic screening for output traits in MMR-defective mutantscan be by biochemical activity and/or a physical phenotype of thealtered gene product. A mutant phenotype can also be detected byidentifying alterations in electrophoretic mobility, DNA binding in thecase of transcription factors, spectroscopic properties such as IR, CD,X-ray crystallography or high field NMR analysis, or other physical orstructural characteristics of a protein encoded by a mutant gene. It isalso possible to screen for altered novel function of a protein in situ,in isolated form, or in model systems. One can screen for alteration ofany property of the microorganism associated with the function of thegene of interest, whether the gene is known prior to the selection orunknown. The aforementioned screening and selection discussion is meantto illustrate the potential means of obtaining novel mutants withcommercially valuable output traits.

[0054] Plasmid expression vectors that harbor the mismatch repair (MMR)gene inserts can be used in combination with a number of commerciallyavailable regulatory sequences to control both the temporal andquantitative biochemical expression level of the dominant negative MMRprotein. The regulatory sequences can be comprised of a promoter,enhancer or promoter/enhancer combination and can be inserted eitherupstream or downstream of the MMR gene to control the expression level.The regulatory promoter sequence can be any of those well known to thosein the art, including but not limited to the lac, tetracycline,tryptophan-inducible, phosphate inducible, T7-polymerase-inducible(Studier et al. (1991) J. Mol. Biol. 219(l):37-44), and steroidinducible constructs as well as sequences which can result in theexcision of the dominant negative mismatch repair gene such as those ofthe Cre-Lox system. These types of regulatory systems have been listedin scientific publications and are familiar to those skilled in the art.

[0055] Once a microorganism with a novel, desired output trait ofinterest is created, the activity of the aberrant MMR activity isattenuated or eliminated by any of a variety of methods, includingremoval of the inducer from the culture medium that is responsible forpromoter activation, gene disruption of the aberrant MMR geneconstructs, electroporation and or chemical curing of the expressionplasmids (Brosius(1988) Biotechnology 10:205-225; Wang et al (1999) J.of Fujian Agricultural University 28:43-46; Fu et. al. (1988) Chem.Abstracts 34:415-418). The expression of the dominant negative MMR genewill be turned on to select for hypermutable microbes with new outputtraits. Next, the expression of the dominant negative dominant negativeMMR allele is rapidly turned off to reconstitute a genetically stablestrain that produces a new output trait of commercial interest. Theresulting microbe is now useful as a stable strain that can be appliedto various commercial applications, depending upon the selection processplaced upon it.

[0056] In cases where genetically deficient mismatch repair bacteria(strains such as but not limited to: M1 (mutS) and in EC2416 (mutS deltaumuDC), and mutL or mutY strains) are used to derive new output traits,transgenic constructs will be used that express wild-type mismatchrepair genes sufficient to complement the genetic defect and thereforerestore mismatch repair activity of the host after trait selection(Grzesiuk et al. (1988) Mutagenesis 13:127-132; Bridges et al. (1997)EMBO J. 16:3349-3356; LeClerc (1996) Science 15:1208-1211; Jaworski, A.et al. (1995) Proc. Natl. Acad. Sci USA 92:11019-11023). The resultingmicrobe is genetically stable and can be applied to various commercialpractices.

[0057] The use of over expressing foreign mismatch repair genes fromhuman and yeast such as human PMS1 (SEQ ID NO:7), human PMS2 (SEQ IDNO:5), mouse PMS2 (SEQ ID NO:3), human MSH2 (SEQ ID NO:9), human MLH1(SEQ ID NO:11), yeast MLH1 (SEQ ID NO:1), human MLH3 (SEQ ID NO:28), aswell as the other homologs identified in other species for these encodedpolypeptides etc.have been previously demonstrated to produce a dominantnegative mutator phenotype in bacterial hosts (Brosh and Matson (1995)J. Bacteriol. 177:5612-5621; Studamire et al. (1998) Mol. Cell. Biol.18:7590-7601; Alani et al. (1997) Mol. Cell. Biol.17:2436-2447). Inaddition, the use of bacterial strains expressing prokaryotic dominantnegative MMR genes as well as hosts that have genomic defects inendogenous MMR proteins have also been previously shown to result in adominant negative mutator phenotype (Strand et al. (1993) Nature365:274-276; Nicolaides et al. (1998) Mol. Cell. Biol. 18:1635-1641).However, the findings disclosed here teach the use of MMR genes,including the human PMSR2 and PMSR3 gene (Nicolaides et al. (1995)Genomics 30:195-206); the related PMS134 truncated MMR gene (Nicolaideset al. (1998) Mol. Cell. Biol. 18:1635-1641); the plant mismatch repairgenes (derived from Arabidopsis thaliana), ATPMS2 (SEQ ID NO:30), AtPMS1 (SEQ ID NO:32), and MutS homolog (SEQ ID NO:34) and those genesthat are homologous to the 134 N-terminal amino acids of the PMS2 genewhich include the MutL family of MMR proteins and including the PMSR andPMS2L homologs described by Hori et al. (PMS2L8 (SEQ ID NO:36) andPMS2L9 (SEQ ID NO:38)) and Nicolaides (Nicolaides et al. (1995) Genomics30:195-206) to create hypermutable microbes. The correspondingpolypeptide sequences for the above-referenced nucleic acid sequencesare as follows: yeast MLH1 (SEQ ID NO:2); mouse PMS2 (SEQ ID NO:4);human PMS2 (SEQ ID NO:6); human PMS1 (SEQ ID NO:8); human MSH2 (SEQ IDNO:10); human MLH1 (SEQ ID NO:12); PMS2-134 (SEQ ID NO:14); human MSH6(SEQ ID NO: 16); human PMSR2 (SEQ ID NO: 18); human PMSR3 (SEQ IDNO:20); human PMSL9 (SEQ ID NO:22); human MLH3 (SEQ ID NO:29); ATPMS2(SEQ ID NO:3 1); ATPMS1 (SEQ ID NO:33); At MutS homolog (SEQ ID NO:35);PMS2L8 (SEQ ID NO:37); and PMS2L9 (SEQ ID NO:39).

[0058] In addition, the invention provides the use of DNA mutagens incombination with MMR defective microbial hosts to enhance thehypermutable production of genetic alterations. This has not beendemonstrated in the art previously as a means to accentuate MMR activityfor generation of microorganisms with clinically relevant output traitssuch as antibiotic resistance.

[0059] In some embodiments of the invention, the bacteria cells arerendered hypermutable by introducing a chemical inhibitor of mismatchrepair into the growth medium. Chemical inhibitors of mismatch repairthat may be used to generate hypermutable bacterial cells includeanthracene-derived compounds comprising the formula:

[0060] In certain preferred embodiments of the invention, the anthracenehas the formula:

[0061] wherein R₁-R₁₀ are independently hydrogen, hydroxyl, amino,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl,N-alkenyl,O-alkynyl, S-alkynyl, N-alkynyl, aryl, substituted aryl,aryloxy, substituted aryloxy, heteroaryl, substituted heteroaryl,aralkyloxy, arylalkyl, alkylaryl, alkylaryloxy, arylsulfonyl,alkylsulfonyl, alkoxycarbonyl, aryloxycarbonyl, guanidino, carboxy, analcohol, an amino acid, sulfonate, alkyl sulfonate, CN, NO₂, an aldehydegroup, an ester, an ether, a crown ether, a ketone, an organosulfurcompound, an organometallic group, a carboxylic acid, an organosiliconor a carbohydrate that optionally contains one or more alkylatedhydroxyl groups;

[0062] wherein said heteroalkyl, heteroaryl, and substituted heteroarylcontain at least one heteroatom that is oxygen, sulfur, a metal atom,phosphorus, silicon or nitrogen; and

[0063] wherein said substituents of said substituted alkyl, substitutedalkenyl, substituted alkynyl, substituted aryl, and substitutedheteroaryl are halogen, CN, NO₂, lower alkyl, aryl, heteroaryl, aralkyl,aralkyloxy, guanidino, alkoxycarbonyl, alkoxy, hydroxy, carboxy andamino;

[0064] and wherein said amino groups optionally substituted with an acylgroup, or 1 to 3 aryl or lower alkyl groups;

[0065] or wherein any two of R₁-R₁₀ can together form a polyether;

[0066] or wherein any two of R₁-R₁₀ can, together with the interveningcarbon atoms of the anthracene core, form a crown ether.

[0067] As used herein, “alkyl” refers to a hydrocarbon containing from 1to about 20 carbon atoms. Alkyl groups may straight, branched, cyclic,or combinations thereof. Alkyl groups thus include, by way ofillustration only, methyl, ethyl, propyl, isopropyl, butyl, isobutyl,cyclopentyl, cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, and thelike. Also included within the definition of “alkyl” are fused and/orpolycyclic aliphatic cyclic ring systems such as, for example,adamantane. As used herein the term “alkenyl” denotes an alkyl grouphaving at least one carbon-carbon double bond. As used herein the term“alkynyl” denotes an alkyl group having at least one carbon-carbontriple bond. In certain preferred embodiments of the invention, theanthracene has the formula:

[0068] wherein R₁-R₁₀ are independently hydrogen, hydroxyl, amino,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl,N-alkenyl,O-alkynyl, S-alkynyl, N-alkynyl, aryl, substituted aryl,aryloxy, substituted aryloxy, heteroaryl, substituted heteroaryl,aralkyloxy, arylalkyl, alkylaryl, alkylaryloxy, arylsulfonyl,alkylsulfonyl, alkoxycarbonyl, aryloxycarbonyl, guanidino, carboxy, analcohol, an amino acid, sulfonate, alkyl sulfonate, CN, NO₂, an aldehydegroup, an ester, an ether, a crown ether, a ketone, an organosulfurcompound, an organometallic group, a carboxylic acid, an organosiliconor a carbohydrate that optionally contains one or more alkylatedhydroxyl groups;

[0069] wherein said heteroalkyl, heteroaryl, and substituted heteroarylcontain at least one heteroatom that is oxygen, sulfur, a metal atom,phosphorus, silicon or nitrogen; and

[0070] wherein said substituents of said substituted alkyl, substitutedalkenyl, substituted alkynyl, substituted aryl, and substitutedheteroaryl are halogen, CN, NO₂, lower alkyl, aryl, heteroaryl, aralkyl,aralkyloxy, guanidino, alkoxycarbonyl, alkoxy, hydroxy, carboxy andamino;

[0071] and wherein said amino groups optionally substituted with an acylgroup, or 1 to 3 aryl or lower alkyl groups;

[0072] or wherein any two of R₁-R₁₀ can together form a polyether;

[0073] or wherein any two of R₁-R₁₀ can, together with the interveningcarbon atoms of the anthracene core, form a crown ether.

[0074] As used herein, “alkyl” refers to a hydrocarbon containing from 1to about 20 carbon atoms. Alkyl groups may straight, branched, cyclic,or combinations thereof. Alkyl groups thus include, by way ofillustration only, methyl, ethyl, propyl, isopropyl, butyl, isobutyl,cyclopentyl, cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, and thelike. Also included within the definition of “alkyl” are fused and/orpolycyclic aliphatic cyclic ring systems such as, for example,adamantane. As used herein the term “alkenyl” denotes an alkyl grouphaving at least one carbon-carbon double bond. As used herein the term“alkynyl” denotes an alkyl group having at least one carbon-carbontriple bond.

[0075] In some embodiments, the anthracene has the formula:

[0076] wherein R₁-R₁₀ are independently hydrogen, hydroxyl, amino,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl,N-alkenyl, O-alkynyl, S-alkynyl, N-alkynyl, aryl, substituted aryl,aryloxy, substituted aryloxy, heteroaryl, substituted heteroaryl,aralkyloxy, arylalkyl, alkylaryl, alkylaryloxy, arylsulfonyl,alkylsulfonyl, alkoxycarbonyl, aryloxycarbonyl, guanidino, carboxy, analcohol, an amino acid, sulfonate, alkyl sulfonate, CN, NO₂, an aldehydegroup, an ester, an ether, a crown ether, a ketone, an organosulfurcompound, an organometallic group, a carboxylic acid, an organosiliconor a carbohydrate that optionally contains one or more alkylatedhydroxyl groups;

[0077] wherein said heteroalkyl, heteroaryl, and substituted heteroarylcontain at least one heteroatom that is oxygen, sulfur, a metal atom,phosphorus, silicon or nitrogen; and

[0078] wherein said substituents of said substituted alkyl, substitutedalkenyl, substituted alkynyl,

[0079] substituted aryl, and substituted heteroaryl are halogen, CN,NO₂, lower alkyl, aryl, heteroaryl, aralkyl, aralkyloxy, guanidino,alkoxycarbonyl, alkoxy, hydroxy, carboxy and amino;

[0080] and wherein said amino groups optionally substituted with an acylgroup, or 1 to 3 aryl or lower alkyl groups.

[0081] Examples of such anthracenes include, but are not limited to1,2-dimethylanthracene, 9,10-dimethyl anthracene,7,8-dimethylanthracene, 9,10-diphenylanthracene,9,10-dihydroxymethylanthracene, 9-hydroxymethyl-10-methylanthracene,dimethylanthracene-1,2-diol,9-hydroxymethyl-10-methylanthracene-1,2-diol,9-hydroxymethyl-10-methylanthracene-3,4-diol, and9,10-di-m-tolyanthracene.

[0082] As used herein the term “anthracene” refers to the compoundanthracene. However, when referred to in the general sense, such as“anthracenes,” “an anthracene” or “the anthracene,” such terms denoteany compound that contains the fused triphenyl core structure ofanthracene, i.e.,

[0083] regardless of extent of substitution.

[0084] In some embodiments, the alkyl, alkenyl, alkynyl, aryl, aryloxy,and heteroaryl substituent groups described above may bear one or morefurther substituent groups; that is, they may be “substituted”. In somepreferred embodiments these substituent groups can include halogens (forexample fluorine, chlorine, bromine and iodine), CN, NO₂, lower alkylgroups, aryl groups, heteroaryl groups, aralkyl groups, aralkyloxygroups, guanidino, alkoxycarbonyl, alkoxy, hydroxy, carboxy and aminogroups. In addition, the alkyl and aryl portions of aralkyloxy,arylalkyl, arylsulfonyl, alkylsulfonyl, alkoxycarbonyl, andaryloxycarbonyl groups also can bear such substituent groups. Thus, byway of example only, substituted alkyl groups include, for example,alkyl groups fluoro-, chloro-, bromo- and iodoalkyl groups, aminoalkylgroups, and hydroxyalkyl groups, such as hydroxymethyl, hydroxyethyl,hydroxypropyl, hydroxybutyl, and the like. In some preferred embodimentssuch hydroxyalkyl groups contain from 1 to about 20 carbons.

[0085] As used herein the term “aryl” means a group having 5 to about 20carbon atoms and which contains at least one aromatic ring, such asphenyl, biphenyl and naphthyl. Preferred aryl groups includeunsubstituted or substituted phenyl and naphthyl groups. The term“aryloxy” denotes an aryl group that is bound through an oxygen atom,for example a phenoxy group.

[0086] In general, the prefix “hetero” denotes the presence of at leastone hetero (i.e., non-carbon) atom, which is in some preferredembodiments independently one to three O, N, S, P, Si or metal atoms.Thus, the term “heteroaryl” denotes an aryl group in which one or morering carbon atom is replaced by such a heteroatom. Preferred heteroarylgroups include pyridyl, pyrimidyl, pyrrolyl, furyl, thienyl, andimidazolyl groups.

[0087] The term “aralkyl” (or “arylalkyl”) is intended to denote a grouphaving from 6 to 15 carbons, consisting of an alkyl group that bears anaryl group. Examples of aralkyl groups include benzyl, phenethyl,benzhydryl and naphthylmethyl groups.

[0088] The term “alkylaryl” (or “alkaryl”) is intended to denote a grouphaving from 6 to 15 carbons, consisting of an aryl group that bears analkyl group. Examples of aralkyl groups include methylphenyl,ethylphenyl and methylnaphthyl groups.

[0089] The term “arylsulfonyl” denotes an aryl group attached through asulfonyl group, for example phenylsulfonyl. The term “alkylsulfonyl”denotes an alkyl group attached through a sulfonyl group, for examplemethylsulfonyl.

[0090] The term “alkoxycarbonyl” denotes a group of formula —C(═O)—O—Rwhere R is alkyl, alkenyl, or aLkynyl, where the alkyl, alkenyl, oralkynyl portions thereof can be optionally substituted as describedherein.

[0091] The term “aryloxycarbonyl” denotes a group of formula —C(═O)—O—Rwhere R is aryl, where the aryl portion thereof can be optionallysubstituted as described herein.

[0092] The terms “arylalkyloxy” or “aralkyloxy” are equivalent, anddenote a group of formula —O—R′—R″, where R′ is R is alkyl, alkenyl, oralkynyl which can be optionally substituted as described herein, andwherein R″ denotes a aryl or substituted aryl group.

[0093] The terms “alkylaryloxy” or “alkaryloxy” are equivalent, anddenote a group of formula —O—R′—R″, where R′ is an aryl or substitutedaryl group, and R″ is alkyl, alkenyl, or alkynyl which can be optionallysubstituted as described herein.

[0094] As used herein, the term “aldehyde group” denotes a group thatbears a moiety of formula —C(═O)—H. The term “ketone” denotes a moietycontaining a group of formula —R—C(═O)—R═, where R and R= areindependently alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, oralkaryl, each of which may be substituted as described herein.

[0095] As used herein, the term “ester” denotes a moiety having a groupof formula —R—C(═O)—O—R= or —R—O—C(═O)—R=where R and R= areindependently alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, oralkaryl, each of which may be substituted as described herein.

[0096] The term “ether” denotes a moiety having a group of formula—R—O—R= or where R and R= are independently alkyl, alkenyl, alkynyl,aryl, heteroaryl, aralkyl, or alkaryl, each of which may be substitutedas described herein.

[0097] The term “crown ether” has its usual meaning of a cyclic ethercontaining several oxygen atoms. As used herein the term “organosulfurcompound” denotes aliphatic or aromatic sulfur containing compounds, forexample thiols and disulfides. The term “organometallic group” denotesan organic molecule containing at least one metal atom.

[0098] The term “organosilicon compound” denotes aliphatic or aromaticsilicon containing compounds, for example alkyl and aryl silanes.

[0099] The term “carboxylic acid” denotes a moiety having a carboxylgroup, other than an amino acid.

[0100] As used herein, the term “amino acid” denotes a moleculecontaining both an amino group and a carboxyl group. In some preferredembodiments, the amino acids are α-, β-, γ- or δ-amino acids, includingtheir stereoisomers and racemates. As used herein the term “L-aminoacid” denotes an a-amino acid having the L configuration around theα-carbon, that is, a carboxylic acid of general formulaCH(COOH)(NH₂)-(side chain), having the L-configuration. The term“D-amino acid” similarly denotes a carboxylic acid of general formulaCH(COOH)(NH₂)-(side chain), having the D-configuration around theα-carbon. Side chains of L-amino acids include naturally occurring andnon-naturally occurring moieties. Non-naturally occurring (i.e.,unnatural) amino acid side chains are moieties that are used in place ofnaturally occurring amino acid side chains in, for example, amino acidanalogs. See, for example, Lehninger, Biochemistry, Second Edition,Worth Publishers, Inc, 1975, pages 72-77, incorporated herein byreference. Amino acid substituents may be attached through theircarbonyl groups through the oxygen or carbonyl carbon thereof, orthrough their amino groups, or through functionalities residing on theirside chain portions.

[0101] In some embodiments of the methods of the invention, the cellsare made hypermutable using ATP analogs capable of blocking ATPaseactivity required for MMR. MMR reporter cells are screened with ATPcompound libraries to identify those compounds capable of blocking MMRin vivo. Examples of ATP analogs that are useful in blocking MMRactivity include, but are not limited to, nonhydrolyzable forms of ATPsuch as AMP-PNP and ATP[gamma]S block the MMR activity (Galio, L. et al.(1999) Nucl. Acids Res. 27:2325-2331; Allen, D. J. et al. (1997) EMBO J.16:4467-4476; Bjornson K. P. et al. (2000) Biochem. 39:3176-3183). TheATPase inhibitors inhibit MMR and the cells become hypermutable as aresult.

[0102] In other embodiments of the methods of the invention, the cellsare made hypermutable using nuclease inhibitors that are able to blockthe exonuclease activity of the MMR biochemical pathway. MMR reportercells are screened with nuclease inhibitor compound libraries toidentify compounds capable of blocking MMR in vivo. Examples of nucleaseinhibitors that are useful in blocking MMR activity include, but are notlimited to analogs of N-Ethylmaleimide, an endonuclease inhibitor(Huang, Y. C., et.al. (1995) Arch. Biochem. Biophys. 316:485),heterodimeric adenine-chain-acridine compounds, exonulcease IIIinhibitors (Belmont P, et.al., Bioorg Med Chem Lett (2000) 10:293-295),as well as antibiotic compounds such as Heliquinomycin, which havehelicase inhibitory activity (Chino, M, et.al. J. Antibiot. (Tokyo)(1998) 51:480-486). The nuclease inhibitors inhibit MMR and the cellsbecome hypermutable as a result.

[0103] In other embodiments of the methods of the invention, the cellsare made hypermutable using DNA polymerase inhibitors that are able toblock the polymerization required for mismatch-mediated repair. MMRreporter cells are screened with DNA polymerase inhibitor compoundlibraries to identify those compounds capable of blocking MMR in vivo.Examples of DNA polymerase inhibitors that are useful in blocking MMRactivity include, but are not limited to, analogs of actinomycin D(Martin, S. J., et.al. (1990) J. Immunol. 145:1859), Aphidicolin(Kuwakado, K. et.al. (1993) Biochem. Pharmacol. 46:1909)1-(2′-Deoxy-2′-fluoro-beta-L-arabinofuranosyl)-5-methyluracil (L-FMAU)(Kukhanova M, et.al., Biochem Pharmacol (1998) 55:1181-1187), and2′,3′-dideoxyribonucleoside 5′-triphosphates (ddNTPs) (Ono, K., et.al.,Biomed Pharmacother (1984) 38:382-389). The polymerase inhibitorsinhibit MMR and the cells become hypermutable as a result.

[0104] Bacterial cells rendered hypermutable using chemical inhibitorsof MMR may be made genetically stable when the desired phenotype isobtained by removing the MMR inhibitory molecule.

[0105] In certain embodiments, the bacterial cells are made hypermutableby introducing plamids that generate antisense messages wherein theantisense RNA specifically bind to MMR genes and prevent efficientexpression of MMR proteins. Preferably, the antisense transcripts are atleast 12 nucleotides in length and, more preferably are at least 20, 30,40, 50 nucleotides or more in length. The antisense transcriptsspecifically bind to regions of the MMR gene to inhibit expression.Preferably, the antisense transcripts specifically bind to regulatoryregions of the MMR gene such as to the MMR promoter region, Kozakconsensus sequences, and the like. As used herein, “specifically bind”refers to association of nucleic acid strands forming complementary basepairing in Watson-Crick arrangement, allowing for mismatches such that100% complementarity is not required. In general, the complementaritywill be about 85%, 90%, 95% or more. Plasmids that may be used toexpress an antisense MMR transcript include any vector generally knownin the art to express antisense transcripts, such as for example, thosefound in Qian Y. et al. (1998) Mutat. Res. 418(2-3):61-71. The abovedisclosure generally describes the present invention. A more completeunderstanding can be obtained by reference to the following specificexamples that will be provided herein for purposes of illustration only,and are not intended to limit the scope of the invention.

EXAMPLE1 Generation of MMR Defective Bacteria

[0106] Bacterial expression constructs were prepared using the humanPMS2 related gene (hPMSR3) (Nicolaides et al. (1995) Genomics30:195-206) and the human PMS134 cDNA (Nicolaides et al. (1998) Mol.Cell. Biol. 18:1635-1641), both of which are capable of inactivating MMRactivity and thereby increase the overall frequency of genomichypermutation. Moreover, the use of regulatable expression vectors willallow for suppression of dominant negative MMR alleles and restorationof the MMR pathway and genetic stability in hosts cells (Brosius, J.(1988) Biotechnology 10:205-225). For these studies, a plasmid encodingthe hPMS134 cDNA was altered by polymerase chain reaction (PCR). The 5′oligonucleotide has the following sequence: 5′-acg cat atg gag cga gctgag agc tcg agt-3′(SEQ ID NO:23) that includes the NdeI restriction site(cat atg). The 3′-oligonucleotide has the following sequence: 5′-gaa ttctta tca cgt aga atc gag acc gag gag agg gtt agg gat agg ctt acc agt tccaac ctt cgc cga tgc-3′ (SEQ ID NO:24) that includes an EcoRi site(gaattc) and the 14 amino acid epitope for the V5 antibody. Theoligonucleotides were used for PCR under standard conditions thatincluded 25 cycles of PCR (95° C. for 1 minute, 55° C. for 1 minute, 72°C. for 1.5 minutes for 25 cycles followed by 3 minutes at 72° C. PCRfragment was purified by gel electrophoresis and cloned into pTA2.1(InVitrogen) by standard cloning methods (Sambrook et al., MOLECULARCLONING: A LABORATORY MANUAL, THIRD EDITION, 2001), creating the plasmidpTA2.1-hPMS134. The pTA2.1-hPMS134 plasmid was digested with therestriction enzyme EcoRi to release the insert (there are two EcoRirestriction sites in the multiple cloning site of pTA2.1 that flank theinsert) and the fragment was end-filled using Klenow fragment and dNTPs.Next, the fragment was gel purified, digested with NdeI, and inserted inpT7-Ea (which had been digested with NdeI and BamHI, end-filledusingKlenow, and phosphatase treated). The new plasmid was designatedpT7-Ea-hPMS134.

[0107] The following strategy, similar to that described above to clonehuman PMS134, was used to construct an expression vector for the humanrelated gene PMSR3. First, the hPMSR3 fragment was amplified by PCR tointroduce two restriction sites: an NdeI restriction site at the 5′-end, and an Eco RI site at the 3′-end of the fragment. The5′-oligonucleotide that was used for PCR has the following sequence:5′-acg cat atg tgt cct tgg cgg cct aga-3′ (SEQ ID NO:25) that includesthe NdeI restriction site (CAT ATG). The 3′-oligonucleotide used for PCRhas the following sequence: 5′-gaa ttc tta tta cgt aga atc gag acc gaggag agg gtt agg gat agg ctt acc cat gtg tga tgt ttc aga gct-3′ (SEQ IDNO:26) that includes an EcoRi site (gaattc) and the V5 epitope to allowfor antibody detection. The plasmid that contained human PMSR3 inpBluescript SK (Nicolaides et al. (1995) Genomics 30:195-206) was usedas the PCR target with the hPMS2-specific oligonucleotides above.Following 25 cycles of PCR (95° C. for 1 minute, 55° C. for 1 minute,72° C for 1.5 minutes for 25 cycles followed by 3 minutes at 72° C.).The PCR fragment was purified by gel electrophoresis and cloned intopTA2.1 (InVitrogen) by standard cloning methods (Sambrook et al.,MOLECULAR CLONING: A LABORATORY MANUAL, THIRD EDITION, 2001), creatingthe plasmid pTA2.1-hR3. The pTA2.1-hR3 plasmid was next digested withthe restriction enzyme EcoRI to release the insert (there are two EcoRIrestriction sites in the multiple cloning site of pTA2.1 that flank theinsert) and the fragment was end-filled using Klenow fragment and dNTPs.Then, the fragment was gel purified, digested with NdeI, and inserted inpT7-Ea (which had been digested with NdeI and BamHI, end-filled usingKlenow, and phosphatase treated). The new plasmid was designatedpT7-Ea-hR3.

[0108] BL21 cells harbor an additional expression vector for thelysozyme protein, which has been demonstrated to bind to the T7polymerase in situ; this results in a bacterial strain that has very lowlevels of T7 polymerase expression. However, upon addition of theinducer isopropyl-beta-D-thiogalactopyranoside (IPTG), the cells expresshigh-levels of T7 polymerase due to the IPTG-inducible element thatdrives expression of the polymerase that is resident within the genomeof the BL21 cells (Studier et al. (1991) J. Mol. Biol. 219(1):37-44).The BL21 cells are chloramphenicol resistant due to the plasmid thatexpresses lysozyme within the cell. To introduce the pT7-hPMS134 or thepT7-hPMSR3 genes into BL21 cells, the cells were made competent byincubating the cells in ice cold 50 mM CaCl₂ for 20 minutes, followed byconcentrating the cells and adding super-coiled plasmid DNA as describe( Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, THIRDEDITION, Cold Spring Harbor Laboratory Press, 2001). Ampicillinresistant BL21 were selected on LB-agar plates [5% yeast extract, 10%bactotryptone, 5% NaCl, 1.5% bactoagar, pH 7.0 (Difco)] platescontaining 25 μg/ml chloramphenicol and 100 μg/ml ampicillin. The nextday, bacterial colonies were selected and analyzed by restrictionendonuclease digestion and sequence analysis for plasmids containing anintact pTACPMS134 or pTAC empty plasmid.

[0109] In addition to constructing a V5-epitope tagged PMS134 construct,we also constructed and tested a non-epitope tagged version. This wasprepared to demonstrate that the epitope tag did not cause thealteration of the dominant-negative phenotype that PMS134 has onmismatch repair activity. For these studies, a BamHI restrictionfragment containing the hPMS134 cDNA was filled-in using Klenow fragmentand then sub-cloned into a Klenow-filled, blunt-ended NdeI-XhoI site ofthe pTACLAC expression vector (which contains the IPTG-induciblebacterial TAC promoter and ampicillin resistance gene as selectablemarker). The NdeI-XhoI cloning site is flanked by the TACLAC promoterthat contains the LacI repressor site followed by a Shine-Dalgarnoribosome-binding site at the 5′ flanking region and the T1T2 ribosomalRNA terminator in the 3′ flanking region. The TACLAC vector alsocontains the LacI gene, which is constitutively expressed by the TACpromoter.

[0110] DH10OB bacterial cells containing the pBCSK vector (Stratagene),which constitutively expresses the, β-galactosidase gene and containsthe chloramphenicol resistance marker for selection, were made competentvia the CaCl₂ method (Sambrook, et al. MOLECULAR CLONING: A LABORATORYMANUAL, Cold Spring Harbor Laboratory Press, 1982). This vector turnsbacterial cells blue when grown in the presence of IPTG and X-gal thataids in the detection of bacterial colonies. Competent cells weretransfected with the pTAC empty vector or the pTACPMS134 vectorfollowing the heat-shock protocol. Transfected cultures were plated ontoLB-agar [5% yeast extract, 10% bactotryptone, 5% NaCl, 1.5% bactoagar,pH 7.0 (Difco)] plates containing 25 μg/ml chloramphenicol and 100 μg/mlampicillin. The next day, bacterial colonies were selected and analyzedby restriction endonuclease digestion and sequence analysis for plasmidscontaining an intact pTACPMS134 or pTAC empty plasmid. Ten clones ofeach bacteria containing correct empty or PMS134 inserts were then grownto confluence overnight in LB media (5% yeast extract, 10%bactotryptone, 5% NaCl, pH 7.0) containing 10 μg/ml chloramphenicol and50 μg/ml ampicillin. The next day TAC empty or pTACPMS134 cultures werediluted 1:4 in LB medium plus 50 μM IPTG (Gold Biotechnology) andcultures were grown for 12 and 24 hours at 37° C. After incubation, 50μl aliquots were taken from each culture and added to 150 μls of 2× SDSbuffer and cultures were analyzed for PMS134 protein expression bywestern blot.

[0111] Western blots were carried out as follows: 50 μls of each PMS134or empty plasmid culture was directly lysed in 2× lysis buffer (60 mMTris, pH 6.8, 2% SDS, 10% glycerol, 0.1 M 2-mercaptoethanol, 0.001%bromophenol blue) and samples were boiled for 5 minutes. Lysate proteinswere separated by electrophoresis on 4-20% Tris glycine gels (Novex).Gels were electroblotted onto Immobilon-P (Millipore) in 48 mM Trisbase, 40 mM glycine, 0.0375% SDS, 20% methanol and blocked overnight at4° C. in Tris-buffered saline plus 0.05% Tween-20 and 5% condensed milk.Filters were probed with a rabbit polyclonal antibody generated againstthe N-terminus of the human PMS2 polypeptide (Santa Cruz), which is ableto recognize the PMS134 polypeptide (Su et al. (1988) J. Biol. Chem.263:6829-6835), followed by a secondary goat anti-rabbit horseradishperoxidase-conjugated antibody. Alternatively, blots were probed with ananti-V5 monoclonal antibody followed by a secondary goat anti-mousehorseradish peroxidase-conjugated antibody. After incubation with thesecondary antibody, blots are developed using chemiluminescence (Pierce)and exposed to film to measure PMS134 expression.

[0112] For induction of PMS gene product, 5 ml cultures of Luria Broth(LB) plus 50 μg/ml ampicillin were inoculated from glycerol stocks ofthe transformants pT7Ea (BL21), pT7PMS134/V5 (BL21), or pT7PMSR3 (BL21)and grown overnight at 37° C. with shaking. 200 μl of each overnightculture was inoculated in 20 ml (1:100) fresh LB broth plus ampicillinand grown to an OD₆₀₀ of 0.6. 20 μl of 100 mM IPTG (final concentration0.1 mM) was added and cultures were grown overnight. Western analysisconfirmed the presence of inducible PMS expression in the presence ofinducer molecule (not shown).

EXAMPLE 2 Generation of Antibiotic Resistant Bacteria

[0113] To demonstrate the ability to produce antibiotic resistantbacterial strains by inhibiting MMR, 10⁷ bacterial cells expressingeither the vector (pT7Ea) or pT7PMS134/V5 were inoculated into 5 ml LBbroth plus the appropriate antibiotic concentrations as shown below(Table 1) and grown overnight at 37° C. with shaking. Antibioticconcentrations were based on 0.5× the minimum inhibitory concentrations(MIC) observed to inhibit the growth of bacteria constitutivelyexpressing the mar operon (Goldman et al. (1996) Antimicrobial AgentsChemother. 40: 1266-1269). Titration analysis found the followingamounts to be effective in inhibiting bacterial growth in the presenceof various compounds. TABLE 1 Half minimum inhibitory concentrations(MIC) on BL21 cells. DRUG 0.5X MIC (μg/ml) Tetracycline 4.70 NalidixicAcid 7.10 Ofloxacin 0.13 Norfloxacin 0.13 Vancomycin 250.0

[0114] The next day, cultures were analyzed for cell growth in thepresence or absence of antibiotics. Table 2 summarizes typical data fromthese studies. No growth was observed in bacterial control cells(pT7Ea), which had OD levels similar to blank culture. In contrast,significant culture growth was observed in pT7PMS134V5 and pT7PMSR3 (notshown) cultures grown in all antibiotics tested (Table 2) TABLE 2Overnight Growth of Drug Resistant Mutants Expressing the PMS2-134.pT7Ea pT7PMS134V5 Drug growth Cell # growth Cell # (X10⁹) Tetracycline −0 + 1.10 Nalidixic Acid − 0 + 0.97 Ofloxacin − 0 + 1.20 Norfloxacin −0 + 1.40 Vancomycin − 0 + ND

[0115] To test the stability of antibiotic resistance, cells werereplated and followed for growth in the presence of 1× MIC concentrationof antibiotic. Table 3 shows an example in which bacterial cells wereinoculated at 1×10⁷ cells/ml and grown for 6 hours in the presence oftetracycline (Tet). As shown in FIG. 1, pT7Ea control culture did notgrow in the presence of Tet while pT7PMS134 and pT7PMSR3 culturesresistant to Tet grew to confluence at time 4 hours after inoculation.These data demonstrate the ability to generate antibiotic resistantcultures by blocking MMR and reestablishing genetically stable culturesthat can be used for gene discovery.

EXAMPLE 3 Genomic Analysis of Antibiotic Resistant Bacteria and TargetDiscovery.

[0116] The ability to generate a wide degree of genomic mutation in MMRdefective bacteria allow for the rapid analysis of the AR host's genomein comparison to the wild-type strain. While many methods for mutationanalysis exist that are know by those skilled in the art, severalapproaches exist that allow for the screening of unknown genes as wellas those that exist which are capable of screening for mutants within“candidate” genes that are capable of conferring an antiobioticresistant phenotype. One such method includes the use of invitro-coupled-translation strategies, which is a rapid method that isused to screen for mutations that result in truncated polypeptides (Liuet al. (1996) Nat. Med. 2:169-174; Nicolaides et al. (1994) Nature 371:75-80; Papadopoulos et al. (1994) Science 263:1625-1629; Nicolaides etal. (1998) Mol. Cell. Biol. 18:1635-1641; Alekshun, M. N. and S. B. Levy(1999) J. Bacteriol. 181:3303-3306).

[0117] In vitro Transcription-coupled-translation

[0118] Linear DNA fragments containing candidate gene sequences wereprepared by PCR, incorporating sequences for in vitro transcription andtranslation in the sense primer. The sense primer contains the leadersequence 5′-tttaatacgactcactatagggagaccaccatggnnn nnn nnn nnn nnn-3′(SEQ ID NO:27) where the series of “n” nuclsotides indicates sequencecorresponding to the first 5 codons. The antisense primer consists ofnucleotide sequences surrounding and including the natural stop codon ofthe gene. DNA fragments are PCR amplified using buffers and condions asdescribed (Nicolaides et al. (1995) Genomics 30:195-206). Two to fivemicroliters of whole bacteria are added to the PCR reaction mix andreactions are carried out at 95° C. for 1 minute for one cycle followedby thirty cycles at 95° C. for 30 sec, 52° C. for 1 minute and 72° C.for 2 minutes. PCR products are then directly added to a rabbitireticulolysate mixture to carry out transcription-coupled-translation(Promega). The reaction mixtures were supplemented with [³⁵S]-methioninefor detection purposes. Translation reactions are incubated for 2 hours.After the reaction is complete, an equal volume of 2× SDS lysis bufferis added to the samples, and the samples are boiled and then loaded onto12% NuPAGE gels (Novex). Gels are run at 150V, dried and exposed toautoradiography. Products that are smaller than the expected molecularweight of the wild-type protein (as compared to the control samples) arethen determined to be mutant and DNA fragments are sequenced to confirmthe presence of a frame-shift/nonsense mutation. This approach has beenused to identify mutations in bacterial genes that have been previouslybeen reported to produce antibiotic resistance in bacteria.

[0119] Discussion

[0120] The results described above lead to several conclusions. Theinhibition of MMR results in an increase in hypermutability in bacteria.This activity is due to the inhibition of MMR biochemical activity inthese hosts. This invention provides a novel method of producingantibiotic resistant strains for target discovery and the rationaldesign of novel anti-microbial agents to each target identified bygenerating AR bacteria through the inhibition of mismatch repair.

[0121] The disclosures of the following references, as well as thereferences cited herein, are hereby incorporated by reference in theirentirety.

[0122] References

[0123] 1. Attfield, P. V. and Pinney, R. J. 1985. Elimination ofmulticopy plasmid R6K by bleomycin. Antimicrob. Agents Chemother.27(6):985-988.

[0124] 2. Baker S. M., Bronner, C. E., Zhang, L., Plug, A. W., Robatez,M., Warren, G., Elliott, E. A., Yu, J., Ashley, T., Arnheim, N.,Bradley, N., Flavell, R. A., and Liskay, R. M. 1995. Male defective inthe DNA mismatch repair gene PMS2 exhibit abnormal chromosome synapsisin meiosis. Cell 82:309-319.

[0125] 3. Berry, A. 1996. Improving production of aromatic compounds inEscherichia coli by metabolic engineering. Trends Biotechnol.14(7):250-256.

[0126] 4. Bronner, C. E., Baker, S. M., Morrison, P. T., Warren, G.,Smith, L. G., Lescoe, M. K., Kane, M., Earabino, C., Lipford, J.,Lindblom, A., Tannergard, P., Bollag, R. J., Godwin, A., R., Ward, D.C., Nordenskjold, M., Fishel, R., Kolodner, R., and Liskay, R. M. 1994.Mutation in the DNA mismatch repair gene homologue hMLH1 is associatedwith hereditary non-polyposis colon cancer. Nature 368:258-261.

[0127] 5. de Wind N., Dekker, M., Berns, A., Radman, M., and Riele, H.T. 1995. Inactivation of the mouse Msh2 gene results in mismatch repairdeficiency, methylation tolerance, hyperrecombination, andpredisposition to cancer. Cell 82:321-300.

[0128] 6. Drummond, J. T., Li, G. M., Longley, M. J., and Modrich, P.1995. Isolation of an hMSH2-p160 heterodimer that restores mismatchrepair to tumor cells. Science 268:1909-1912.

[0129] 7. Drummond, J. T., Anthoney, A., Brown, R., and Modrich, P.1996. Cisplatin and adriamycin resistance are associated with MutLa andmismatch repair deficiency in an ovarian tumor cell line. J.Biol.Chem.271:9645-19648.

[0130] 8. Edelmann, W., Cohen, P. E., Kane, M., Lau, K., Morrow, B.,Bennett, S., Umar, A., Kunkel, T., Cattoretti, G., Chagnatti, R.,Pollard, J. W., Kolodner, R. D., and Kucherlapati, R. 1996. Meioticpachytene arrest in MLH1-deficient mice. Cell 85:1125-1134.

[0131] 9. Fishel, R., Lescoe, M., Rao, M. R. S., Copeland, N. J.,Jenkins, N. A., Garber, J., Kane, M., and Kolodner, R. 1993. The humanmutator gene homolog MSH2 and its association with hereditarynonpolyposis colon cancer. Cell 7:1027-1038.

[0132] 10. Fu, K. P., Grace, M. E., Hsiao, C. L., and Hung, P. P. 1988.Elinination of antibiotic-resistant plasmids by quinolone antibiotics.Chemotherapy 34(5):415-418.

[0133] 11. Leach, F. S., Nicolaides, N. C, Papadopoulos, N., Liu, B.,Jen, J., Parsons, R., Peltomaki, P., Sistonen, P., Aaltonen, L. A.,Nystrom-Lahti, M., Guan, X. Y., Zhang, J., Meltzer, P. S., Yu, J. W.,Kao, F. T., Chen, D. J., Cerosaletti, K. M., Fournier, R. E. K., Todd,S., Lewis, T., Leach R. J., Naylor, S. L., Weissenbach, J., Mecklin, J.P., Jarvinen, J. A., Petersen, G. M., Hamilton, S. R., Green, J., Jass,J., Watson, P., Lynch, H. T., Trent, J. M., de la Chapelle, A., Kinzler,K. W., and Vogelstein, B. 1993. Mutations of a mutS homolog inhereditary non-polyposis colorectal cancer. Cell 75:1215-1225.

[0134] 12. Li, G. -M., and Modrich, P. 1995. Restoration of mismatchrepair to nuclear extracts of H6 colorectal tumor cells by a heterodimerof human mutL homologs. Proc. Natl. Acad. Sci. USA 92:1950-1954.

[0135] 13. Liu, B., Parsons, R., Papadopoulos, N., Nicolaides, N. C.,Lynch, H. T., Watson, P., Jass, J. R., Dunlop, M., Wyllie, A.,Peltomaki, P., de la Chapelle, A., Hamilton, S. R., Vogelstein, B., andKinzler, K. W. 1996. Analysis of mismatch repair genes in hereditarynon-polyposis colorectal cancer patients. Nat. Med. 2:169-174.

[0136] 14. Modrich, P. 1994. Mismatch repair, genetic stability, andcancer. Science 266:1959-1960.

[0137] 15. Nicolaides, N. C., Gualdi, R., Casadevall, C., Manzella, L.,and Calabretta, B. 1991. Positive autoregulation of c-myb expression viaMYB binding in the 5′ flanking region of the human c-myb gene. Mol.Cell. Biol. 11:6166-6176.

[0138] 16. Nicolaides, N. C., Correa, I., Casadevall, C., Travali, S.,Soprano, K. J., and Calabretta, B. 1992. The Jun family members, c-JUNand JUND, transactivate the human c-myb promoter via an Ap1 likeelement. J. Biol. Chem. 267, 19665-19672.

[0139] 17. Nicolaides, N. C., Papadopoulos, N., Liu, B., Wei, Y. F.,Carter, K. C., Ruben, S. M., Rosen, C. A., Haseltine, W. A.,Fleischmann, R. D., Fraser, C. M., Adams, M. D., Venter, C. J., Dunlop,M. G., Hamilton, S. R., Petersen, G. M., de la Chapelle, A., Vogelstein,B., and kinzler, K. W. 1994. Mutations of two PMS homologs in hereditarynonpolyposis colon cancer. Nature 371: 75-80.

[0140] 18. Nicolaides N. C., Kinzler, K. W., and Vogelstein, B. 1995.Analysis of the 5′ region of PMS2 reveals heterogenous transcripts and anovel overlapping gene. Genomics 29:329-334.

[0141] 19. Nicolaides, N. C., Carter, K. C., Shell, B. K., Papadopoulos,N., Vogelstein, B., and Kinzler, K. W. 1995. Genomic organization of thehuman PMS2 gene family. Genomics 30:195-206.

[0142] 20. Nicolaides, N. C., Palombo, F., Kinzler, K. W., Vogelstein,B., and Jiricny, J. 1996. Molecular cloning of the N-terminus of GTBP.Genomics 31:395-397.

[0143] 21. Palombo, F., Hughes, M., Jiricny, J., Truong, O., Hsuan, J.1994. Mismatch repair and cancer. Nature 36:417.

[0144] 22. Palombo, F., Gallinari, P., Iaccarino, I., Lettieri, T.,Hughes, M. A., Truong, O., Hsuan, J. J., and Jiricny, J. 1995. GTBP, a160-kilodalton protein essential for mismatch-binding activity in humancells. Science 268:1912-1914.

[0145] 23. Pang, Q., Prolla, T. A., and Liskay, R. M. 1997. Functionaldomains of the Saccharomyces cerevisiae Mlh1p and Pms1p DNA mismatchrepair proteins and their relevance to human hereditary nonpolyposiscolorectal cancer-associated mutations. Mol. Cell. Biol. 17:4465-4473.

[0146] 24. Papadopoulos, N., Nicolaides, N. C., Wei, Y. F., Carter, K.C., Ruben, S. M., Rosen, C. A., Haseltine, W. A., Fleischmann, R. D.,Fraser, C. M., Adams, M. D., Venter, C. J., Dunlop, M. G., Hamilton, S.R., Petersen, G. M., de la Chapelle, A., Vogelstein, B., and kinzler, K.W. 1994. Mutation of a mutL homolog is associated with hereditary coloncancer. Science 263:1625-1629.

[0147] 25. Parsons, R., Li, G. M., Longley, M. J., Fang, W. H.,Papadopolous, N., Jen, J., de la Chapelle, A., Kinzler, K. W.,Vogelstein, B., and Modrich, P. 1993. Hypermutability and mismatchrepair deficiency in RER⁺tumor cells. Cell 75:1227-1236.

[0148] 26. Parsons, R., Li, G. M., Longley, M., Modrich, P., Liu, B.,Berk, T., Hamilton, S. R., Kinzler, K. W., and Vogelstein, B. 1995.Mismatch repair deficiency in phenotypically normal human cells. Science268:738-740.

[0149] 27. Perucho, M. 1996. Cancer of the microsattelite mutatorphenotype. Biol Chem. 377:675-684.

[0150] 28. Prolla, T. A, Pang, Q., Alani, E., Kolodner, R. A., andLiskay, R. M. 1994. MLH1, PMS1, and MSH2 Interaction during theinitiation of DNA mismatch repair in yeast. Science 264:1091-1093.

[0151] 29. Strand, M., Prolla, T. A., Liskay, R. M., and Petes, T. D.Destabilization of tracts of simple repetitive DNA in yeast by mutationsaffecting DNA mismatch repair. 1993. Nature 365:274-276.

[0152] 30. Studier, F. W. Use of bacteriophage T7 lysozyme to improve aninducible T7 expression system. 1991. J. Mol. Biol. 219(1):37-44.

[0153] 31. Su, S. S., Lahue, R. S., Au, K. G., and Modrich, P. 1988.Mispair specificity of methyl directed DNA mismatch corrections invitro. J. Biol. Chem. 263:6829-6835.

[0154] 32. Nicolaides N C, Littnan S J, Modrich P, Kinzler K W,Vogelstein B 1998. A naturally occurring hPMS2 mutation can confer adominant negative mutator phenotype. Mol Cell Biol 18:1635-1641.

[0155] 33. Aronshtam A, Marinus M G. 1996. Dominant negative mutatormutations in the mutL gene of Escherichia coli. Nucleic Acids Res24:2498-2504.

[0156] 34. Wu T H, Marinus M G. 1994. Dominant negative mutatormutations in the mutS gene of Escherichia coli. J Bacteriol176:5393-400.

[0157] 35. Brosh R M Jr, Matson S W. 1995. Mutations in motif II ofEscherichia coli DNA helicase II render the enzyme nonfunctional in bothmismatch repair and excision repair with differential effects on theunwinding reaction. J Bacteriol 177:5612-5621.

[0158] 36. Studamire B, Quach T, Alani, E. 1998. Saccharomycescerevisiae Msh2p and Msh6p ATPase activities are both required duringmismatch repair. Mol Cell Biol 18:7590-7601.

[0159] 37. Alani E, Sokolsky T, Studamire B, Miret J J, Lahue RS. 1997.Genetic and biochemical analysis of Msh2p-Msh6p: role of ATP hydrolysisand Msh2p-Msh6p subunit interactions in mismatch base pair recognition.Mol Cell Biol 17:2436-2447.

[0160] 38. Lipkin S M, Wang V, Jacoby R, Banerjee-Basu S, Baxevanis A D,Lynch H T, Elliott R M, and Collins F S. 2000. MLH3: a DNA mismatchrepair gene associated with mammalian microsatellite instability. NatGenet 24:27-35.

[0161] 39. Lee C C, Lin H K, Lin J K. 1994. A reverse mutagenicity assayfor alkylating agents based on a point mutation in the beta-lactamasegene at the active site serine codon. Mutagenesis 9:401-405.

[0162] 40. Vidal A, Abril N, Pueyo C. 1995. DNA repair by Ogtalkyltransferase influences EMS mutational specificity. Carcinogenesis16:817-821.

[0163] 41. Fu, K. P., Grace, M. E., Hsiao, C. L., and Hung, P. P. 1988.Elimination of antibiotic-resistant plasmids by quinolone antibiotics.Chem. Abstracts 34(5):415-418.

[0164] 42. BiWang, H., ZhiPeng, H., and Xiong, G. 1999. Transformationof Escherichia coli and Bacillus thuringiensis and their plasmid curingby electroporation. J. of Fujian Agricultural University 28(1):43-46.

[0165] 43. Brosius, J. 1988. Expression vectors employing lambda-, trp-,lac-, and lpp-derived promoters. Biotechnology 10:205-225.

[0166] 44. Alekshun, M. N. and Levy, S. B. 1999. Characterization ofMarR Superrepressor Mutants. J. Bacteriol. 181:3303-3306.

1 39 1 3218 DNA Saccharomyces cerevisiae 1 aaataggaat gtgataccttctattgcatg caaagatagt gtaggaggcg ctgctattgc 60 caaagacttt tgagaccgcttgctgtttca ttatagttga ggagttctcg aagacgagaa 120 attagcagtt ttcggtgtttagtaatcgcg ctagcatgct aggacaattt aactgcaaaa 180 ttttgatacg atagtgatagtaaatggaag gtaaaaataa catagaccta tcaataagca 240 atgtctctca gaataaaagcacttgatgca tcagtggtta acaaaattgc tgcaggtgag 300 atcataatat cccccgtaaatgctctcaaa gaaatgatgg agaattccat cgatgcgaat 360 gctacaatga ttgatattctagtcaaggaa ggaggaatta aggtacttca aataacagat 420 aacggatctg gaattaataaagcagacctg ccaatcttat gtgagcgatt cacgacgtcc 480 aaattacaaa aattcgaagatttgagtcag attcaaacgt atggattccg aggagaagct 540 ttagccagta tctcacatgtggcaagagtc acagtaacga caaaagttaa agaagacaga 600 tgtgcatgga gagtttcatatgcagaaggt aagatgttgg aaagccccaa acctgttgct 660 ggaaaagacg gtaccacgatcctagttgaa gacctttttt tcaatattcc ttctagatta 720 agggccttga ggtcccataatgatgaatac tctaaaatat tagatgttgt cgggcgatac 780 gccattcatt ccaaggacattggcttttct tgtaaaaagt tcggagactc taattattct 840 ttatcagtta aaccttcatatacagtccag gataggatta ggactgtgtt caataaatct 900 gtggcttcga atttaattacttttcatatc agcaaagtag aagatttaaa cctggaaagc 960 gttgatggaa aggtgtgtaatttgaatttc atatccaaaa agtccatttc attaattttt 1020 ttcattaata atagactagtgacatgtgat cttctaagaa gagctttgaa cagcgtttac 1080 tccaattatc tgccaaagggcttcagacct tttatttatt tgggaattgt tatagatccg 1140 gcggctgttg atgttaacgttcacccgaca aagagagagg ttcgtttcct gagccaagat 1200 gagatcatag agaaaatcgccaatcaattg cacgccgaat tatctgccat tgatacttca 1260 cgtactttca aggcttcttcaatttcaaca aacaagccag agtcattgat accatttaat 1320 gacaccatag aaagtgataggaataggaag agtctccgac aagcccaagt ggtagagaat 1380 tcatatacga cagccaatagtcaactaagg aaagcgaaaa gacaagagaa taaactagtc 1440 agaatagatg cttcacaagctaaaattacg tcatttttat cctcaagtca acagttcaac 1500 tttgaaggat cgtctacaaagcgacaactg agtgaaccca aggtaacaaa tgtaagccac 1560 tcccaagagg cagaaaagctgacactaaat gaaagcgaac aaccgcgtga tgccaataca 1620 atcaatgata atgacttgaaggatcaacct aagaagaaac aaaagttggg ggattataaa 1680 gttccaagca ttgccgatgacgaaaagaat gcactcccga tttcaaaaga cgggtatatt 1740 agagtaccta aggagcgagttaatgttaat cttacgagta tcaagaaatt gcgtgaaaaa 1800 gtagatgatt cgatacatcgagaactaaca gacatttttg caaatttgaa ttacgttggg 1860 gttgtagatg aggaaagaagattagccgct attcagcatg acttaaagct ttttttaata 1920 gattacggat ctgtgtgctatgagctattc tatcagattg gtttgacaga cttcgcaaac 1980 tttggtaaga taaacctacagagtacaaat gtgtcagatg atatagtttt gtataatctc 2040 ctatcagaat ttgacgagttaaatgacgat gcttccaaag aaaaaataat tagtaaaata 2100 tgggacatga gcagtatgctaaatgagtac tattccatag aattggtgaa tgatggtcta 2160 gataatgact taaagtctgtgaagctaaaa tctctaccac tacttttaaa aggctacatt 2220 ccatctctgg tcaagttaccattttttata tatcgcctgg gtaaagaagt tgattgggag 2280 gatgaacaag agtgtctagatggtatttta agagagattg cattactcta tatacctgat 2340 atggttccga aagtcgatacactcgatgca tcgttgtcag aagacgaaaa agcccagttt 2400 ataaatagaa aggaacacatatcctcatta ctagaacacg ttctcttccc ttgtatcaaa 2460 cgaaggttcc tggcccctagacacattctc aaggatgtcg tggaaatagc caaccttcca 2520 gatctataca aagtttttgagaggtgttaa ctttaaaacg ttttggctgt aataccaaag 2580 tttttgttta tttcctgagtgtgattgtgt ttcatttgaa agtgtatgcc ctttccttta 2640 acgattcatc cgcgagatttcaaaggatat gaaatatggt tgcagttagg aaagtatgtc 2700 agaaatgtat attcggattgaaactcttct aatagttctg aagtcacttg gttccgtatt 2760 gttttcgtcc tcttcctcaagcaacgattc ttgtctaagc ttattcaacg gtaccaaaga 2820 cccgagtcct tttatgagagaaaacatttc atcatttttc aactcaatta tcttaatatc 2880 attttgtagt attttgaaaacaggatggta aaacgaatca cctgaatcta gaagctgtac 2940 cttgtcccat aaaagttttaatttactgag cctttcggtc aagtaaacta gtttatctag 3000 ttttgaaccg aatattgtgggcagatttgc agtaagttca gttagatcta ctaaaagttg 3060 tttgacagca gccgattccacaaaaatttg gtaaaaggag atgaaagaga cctcgcgcgt 3120 aatggtttgc atcaccatcggatgtctgtt gaaaaactca ctttttgcat ggaagttatt 3180 aacaataaga ctaatgattaccttagaata atgtataa 3218 2 769 PRT Saccharomyces cerevisiae 2 Met SerLeu Arg Ile Lys Ala Leu Asp Ala Ser Val Val Asn Lys Ile 1 5 10 15 AlaAla Gly Glu Ile Ile Ile Ser Pro Val Asn Ala Leu Lys Glu Met 20 25 30 MetGlu Asn Ser Ile Asp Ala Asn Ala Thr Met Ile Asp Ile Leu Val 35 40 45 LysGlu Gly Gly Ile Lys Val Leu Gln Ile Thr Asp Asn Gly Ser Gly 50 55 60 IleAsn Lys Ala Asp Leu Pro Ile Leu Cys Glu Arg Phe Thr Thr Ser 65 70 75 80Lys Leu Gln Lys Phe Glu Asp Leu Ser Gln Ile Gln Thr Tyr Gly Phe 85 90 95Arg Gly Glu Ala Leu Ala Ser Ile Ser His Val Ala Arg Val Thr Val 100 105110 Thr Thr Lys Val Lys Glu Asp Arg Cys Ala Trp Arg Val Ser Tyr Ala 115120 125 Glu Gly Lys Met Leu Glu Ser Pro Lys Pro Val Ala Gly Lys Asp Gly130 135 140 Thr Thr Ile Leu Val Glu Asp Leu Phe Phe Asn Ile Pro Ser ArgLeu 145 150 155 160 Arg Ala Leu Arg Ser His Asn Asp Glu Tyr Ser Lys IleLeu Asp Val 165 170 175 Val Gly Arg Tyr Ala Ile His Ser Lys Asp Ile GlyPhe Ser Cys Lys 180 185 190 Lys Phe Gly Asp Ser Asn Tyr Ser Leu Ser ValLys Pro Ser Tyr Thr 195 200 205 Val Gln Asp Arg Ile Arg Thr Val Phe AsnLys Ser Val Ala Ser Asn 210 215 220 Leu Ile Thr Phe His Ile Ser Lys ValGlu Asp Leu Asn Leu Glu Ser 225 230 235 240 Val Asp Gly Lys Val Cys AsnLeu Asn Phe Ile Ser Lys Lys Ser Ile 245 250 255 Ser Leu Ile Phe Phe IleAsn Asn Arg Leu Val Thr Cys Asp Leu Leu 260 265 270 Arg Arg Ala Leu AsnSer Val Tyr Ser Asn Tyr Leu Pro Lys Gly Phe 275 280 285 Arg Pro Phe IleTyr Leu Gly Ile Val Ile Asp Pro Ala Ala Val Asp 290 295 300 Val Asn ValHis Pro Thr Lys Arg Glu Val Arg Phe Leu Ser Gln Asp 305 310 315 320 GluIle Ile Glu Lys Ile Ala Asn Gln Leu His Ala Glu Leu Ser Ala 325 330 335Ile Asp Thr Ser Arg Thr Phe Lys Ala Ser Ser Ile Ser Thr Asn Lys 340 345350 Pro Glu Ser Leu Ile Pro Phe Asn Asp Thr Ile Glu Ser Asp Arg Asn 355360 365 Arg Lys Ser Leu Arg Gln Ala Gln Val Val Glu Asn Ser Tyr Thr Thr370 375 380 Ala Asn Ser Gln Leu Arg Lys Ala Lys Arg Gln Glu Asn Lys LeuVal 385 390 395 400 Arg Ile Asp Ala Ser Gln Ala Lys Ile Thr Ser Phe LeuSer Ser Ser 405 410 415 Gln Gln Phe Asn Phe Glu Gly Ser Ser Thr Lys ArgGln Leu Ser Glu 420 425 430 Pro Lys Val Thr Asn Val Ser His Ser Gln GluAla Glu Lys Leu Thr 435 440 445 Leu Asn Glu Ser Glu Gln Pro Arg Asp AlaAsn Thr Ile Asn Asp Asn 450 455 460 Asp Leu Lys Asp Gln Pro Lys Lys LysGln Lys Leu Gly Asp Tyr Lys 465 470 475 480 Val Pro Ser Ile Ala Asp AspGlu Lys Asn Ala Leu Pro Ile Ser Lys 485 490 495 Asp Gly Tyr Ile Arg ValPro Lys Glu Arg Val Asn Val Asn Leu Thr 500 505 510 Ser Ile Lys Lys LeuArg Glu Lys Val Asp Asp Ser Ile His Arg Glu 515 520 525 Leu Thr Asp IlePhe Ala Asn Leu Asn Tyr Val Gly Val Val Asp Glu 530 535 540 Glu Arg ArgLeu Ala Ala Ile Gln His Asp Leu Lys Leu Phe Leu Ile 545 550 555 560 AspTyr Gly Ser Val Cys Tyr Glu Leu Phe Tyr Gln Ile Gly Leu Thr 565 570 575Asp Phe Ala Asn Phe Gly Lys Ile Asn Leu Gln Ser Thr Asn Val Ser 580 585590 Asp Asp Ile Val Leu Tyr Asn Leu Leu Ser Glu Phe Asp Glu Leu Asn 595600 605 Asp Asp Ala Ser Lys Glu Lys Ile Ile Ser Lys Ile Trp Asp Met Ser610 615 620 Ser Met Leu Asn Glu Tyr Tyr Ser Ile Glu Leu Val Asn Asp GlyLeu 625 630 635 640 Asp Asn Asp Leu Lys Ser Val Lys Leu Lys Ser Leu ProLeu Leu Leu 645 650 655 Lys Gly Tyr Ile Pro Ser Leu Val Lys Leu Pro PhePhe Ile Tyr Arg 660 665 670 Leu Gly Lys Glu Val Asp Trp Glu Asp Glu GlnGlu Cys Leu Asp Gly 675 680 685 Ile Leu Arg Glu Ile Ala Leu Leu Tyr IlePro Asp Met Val Pro Lys 690 695 700 Val Asp Thr Leu Asp Ala Ser Leu SerGlu Asp Glu Lys Ala Gln Phe 705 710 715 720 Ile Asn Arg Lys Glu His IleSer Ser Leu Leu Glu His Val Leu Phe 725 730 735 Pro Cys Ile Lys Arg ArgPhe Leu Ala Pro Arg His Ile Leu Lys Asp 740 745 750 Val Val Glu Ile AlaAsn Leu Pro Asp Leu Tyr Lys Val Phe Glu Arg 755 760 765 Cys 3 3056 DNAMus musculus 3 gaattccggt gaaggtcctg aagaatttcc agattcctga gtatcattggaggagacaga 60 taacctgtcg tcaggtaacg atggtgtata tgcaacagaa atgggtgttcctggagacgc 120 gtcttttccc gagagcggca ccgcaactct cccgcggtga ctgtgactggaggagtcctg 180 catccatgga gcaaaccgaa ggcgtgagta cagaatgtgc taaggccatcaagcctattg 240 atgggaagtc agtccatcaa atttgttctg ggcaggtgat actcagtttaagcaccgctg 300 tgaaggagtt gatagaaaat agtgtagatg ctggtgctac tactattgatctaaggctta 360 aagactatgg ggtggacctc attgaagttt cagacaatgg atgtggggtagaagaagaaa 420 actttgaagg tctagctctg aaacatcaca catctaagat tcaagagtttgccgacctca 480 cgcaggttga aactttcggc tttcgggggg aagctctgag ctctctgtgtgcactaagtg 540 atgtcactat atctacctgc cacgggtctg caagcgttgg gactcgactggtgtttgacc 600 ataatgggaa aatcacccag aaaactccct acccccgacc taaaggaaccacagtcagtg 660 tgcagcactt attttataca ctacccgtgc gttacaaaga gtttcagaggaacattaaaa 720 aggagtattc caaaatggtg caggtcttac aggcgtactg tatcatctcagcaggcgtcc 780 gtgtaagctg cactaatcag ctcggacagg ggaagcggca cgctgtggtgtgcacaagcg 840 gcacgtctgg catgaaggaa aatatcgggt ctgtgtttgg ccagaagcagttgcaaagcc 900 tcattccttt tgttcagctg ccccctagtg acgctgtgtg tgaagagtacggcctgagca 960 cttcaggacg ccacaaaacc ttttctacgt ttcgggcttc atttcacagtgcacgcacgg 1020 cgccgggagg agtgcaacag acaggcagtt tttcttcatc aatcagaggccctgtgaccc 1080 agcaaaggtc tctaagcttg tcaatgaggt tttatcacat gtataaccggcatcagtacc 1140 catttgtcgt ccttaacgtt tccgttgact cagaatgtgt ggatattaatgtaactccag 1200 ataaaaggca aattctacta caagaagaga agctattgct ggccgttttaaagacctcct 1260 tgataggaat gtttgacagt gatgcaaaca agcttaatgt caaccagcagccactgctag 1320 atgttgaagg taacttagta aagctgcata ctgcagaact agaaaagcctgtgccaggaa 1380 agcaagataa ctctccttca ctgaagagca cagcagacga gaaaagggtagcatccatct 1440 ccaggctgag agaggccttt tctcttcatc ctactaaaga gatcaagtctaggggtccag 1500 agactgctga actgacacgg agttttccaa gtgagaaaag gggcgtgttatcctcttatc 1560 cttcagacgt catctcttac agaggcctcc gtggctcgca ggacaaattggtgagtccca 1620 cggacagccc tggtgactgt atggacagag agaaaataga aaaagactcagggctcagca 1680 gcacctcagc tggctctgag gaagagttca gcaccccaga agtggccagtagctttagca 1740 gtgactataa cgtgagctcc ctagaagaca gaccttctca ggaaaccataaactgtggtg 1800 acctggactg ccgtcctcca ggtacaggac agtccttgaa gccagaagaccatggatatc 1860 aatgcaaagc tctacctcta gctcgtctgt cacccacaaa tgccaagcgcttcaagacag 1920 aggaaagacc ctcaaatgtc aacatttctc aaagattgcc tggtcctcagagcacctcag 1980 cagctgaggt cgatgtagcc ataaaaatga ataagagaat cgtgctcctcgagttctctc 2040 tgagttctct agctaagcga atgaagcagt tacagcacct aaaggcgcagaacaaacatg 2100 aactgagtta cagaaaattt agggccaaga tttgccctgg agaaaaccaagcagcagaag 2160 atgaactcag aaaagagatt agtaaatcga tgtttgcaga gatggagatcttgggtcagt 2220 ttaacctggg atttatagta accaaactga aagaggacct cttcctggtggaccagcatg 2280 ctgcggatga gaagtacaac tttgagatgc tgcagcagca cacggtgctccaggcgcaga 2340 ggctcatcac accccagact ctgaacttaa ctgctgtcaa tgaagctgtactgatagaaa 2400 atctggaaat attcagaaag aatggctttg actttgtcat tgatgaggatgctccagtca 2460 ctgaaagggc taaattgatt tccttaccaa ctagtaaaaa ctggacctttggaccccaag 2520 atatagatga actgatcttt atgttaagtg acagccctgg ggtcatgtgccggccctcac 2580 gagtcagaca gatgtttgct tccagagcct gtcggaagtc agtgatgattggaacggcgc 2640 tcaatgcgag cgagatgaag aagctcatca cccacatggg tgagatggaccacccctgga 2700 actgccccca cggcaggcca accatgaggc acgttgccaa tctggatgtcatctctcaga 2760 actgacacac cccttgtagc atagagttta ttacagattg ttcggtttgcaaagagaagg 2820 ttttaagtaa tctgattatc gttgtacaaa aattagcatg ctgctttaatgtactggatc 2880 catttaaaag cagtgttaag gcaggcatga tggagtgttc ctctagctcagctacttggg 2940 tgatccggtg ggagctcatg tgagcccagg actttgagac cactccgagccacattcatg 3000 agactcaatt caaggacaaa aaaaaaaaga tatttttgaa gccttttaaaaaaaaa 3056 4 859 PRT Mus musculus 4 Met Glu Gln Thr Glu Gly Val Ser ThrGlu Cys Ala Lys Ala Ile Lys 1 5 10 15 Pro Ile Asp Gly Lys Ser Val HisGln Ile Cys Ser Gly Gln Val Ile 20 25 30 Leu Ser Leu Ser Thr Ala Val LysGlu Leu Ile Glu Asn Ser Val Asp 35 40 45 Ala Gly Ala Thr Thr Ile Asp LeuArg Leu Lys Asp Tyr Gly Val Asp 50 55 60 Leu Ile Glu Val Ser Asp Asn GlyCys Gly Val Glu Glu Glu Asn Phe 65 70 75 80 Glu Gly Leu Ala Leu Lys HisHis Thr Ser Lys Ile Gln Glu Phe Ala 85 90 95 Asp Leu Thr Gln Val Glu ThrPhe Gly Phe Arg Gly Glu Ala Leu Ser 100 105 110 Ser Leu Cys Ala Leu SerAsp Val Thr Ile Ser Thr Cys His Gly Ser 115 120 125 Ala Ser Val Gly ThrArg Leu Val Phe Asp His Asn Gly Lys Ile Thr 130 135 140 Gln Lys Thr ProTyr Pro Arg Pro Lys Gly Thr Thr Val Ser Val Gln 145 150 155 160 His LeuPhe Tyr Thr Leu Pro Val Arg Tyr Lys Glu Phe Gln Arg Asn 165 170 175 IleLys Lys Glu Tyr Ser Lys Met Val Gln Val Leu Gln Ala Tyr Cys 180 185 190Ile Ile Ser Ala Gly Val Arg Val Ser Cys Thr Asn Gln Leu Gly Gln 195 200205 Gly Lys Arg His Ala Val Val Cys Thr Ser Gly Thr Ser Gly Met Lys 210215 220 Glu Asn Ile Gly Ser Val Phe Gly Gln Lys Gln Leu Gln Ser Leu Ile225 230 235 240 Pro Phe Val Gln Leu Pro Pro Ser Asp Ala Val Cys Glu GluTyr Gly 245 250 255 Leu Ser Thr Ser Gly Arg His Lys Thr Phe Ser Thr PheArg Ala Ser 260 265 270 Phe His Ser Ala Arg Thr Ala Pro Gly Gly Val GlnGln Thr Gly Ser 275 280 285 Phe Ser Ser Ser Ile Arg Gly Pro Val Thr GlnGln Arg Ser Leu Ser 290 295 300 Leu Ser Met Arg Phe Tyr His Met Tyr AsnArg His Gln Tyr Pro Phe 305 310 315 320 Val Val Leu Asn Val Ser Val AspSer Glu Cys Val Asp Ile Asn Val 325 330 335 Thr Pro Asp Lys Arg Gln IleLeu Leu Gln Glu Glu Lys Leu Leu Leu 340 345 350 Ala Val Leu Lys Thr SerLeu Ile Gly Met Phe Asp Ser Asp Ala Asn 355 360 365 Lys Leu Asn Val AsnGln Gln Pro Leu Leu Asp Val Glu Gly Asn Leu 370 375 380 Val Lys Leu HisThr Ala Glu Leu Glu Lys Pro Val Pro Gly Lys Gln 385 390 395 400 Asp AsnSer Pro Ser Leu Lys Ser Thr Ala Asp Glu Lys Arg Val Ala 405 410 415 SerIle Ser Arg Leu Arg Glu Ala Phe Ser Leu His Pro Thr Lys Glu 420 425 430Ile Lys Ser Arg Gly Pro Glu Thr Ala Glu Leu Thr Arg Ser Phe Pro 435 440445 Ser Glu Lys Arg Gly Val Leu Ser Ser Tyr Pro Ser Asp Val Ile Ser 450455 460 Tyr Arg Gly Leu Arg Gly Ser Gln Asp Lys Leu Val Ser Pro Thr Asp465 470 475 480 Ser Pro Gly Asp Cys Met Asp Arg Glu Lys Ile Glu Lys AspSer Gly 485 490 495 Leu Ser Ser Thr Ser Ala Gly Ser Glu Glu Glu Phe SerThr Pro Glu 500 505 510 Val Ala Ser Ser Phe Ser Ser Asp Tyr Asn Val SerSer Leu Glu Asp 515 520 525 Arg Pro Ser Gln Glu Thr Ile Asn Cys Gly AspLeu Asp Cys Arg Pro 530 535 540 Pro Gly Thr Gly Gln Ser Leu Lys Pro GluAsp His Gly Tyr Gln Cys 545 550 555 560 Lys Ala Leu Pro Leu Ala Arg LeuSer Pro Thr Asn Ala Lys Arg Phe 565 570 575 Lys Thr Glu Glu Arg Pro SerAsn Val Asn Ile Ser Gln Arg Leu Pro 580 585 590 Gly Pro Gln Ser Thr SerAla Ala Glu Val Asp Val Ala Ile Lys Met 595 600 605 Asn Lys Arg Ile ValLeu Leu Glu Phe Ser Leu Ser Ser Leu Ala Lys 610 615 620 Arg Met Lys GlnLeu Gln His Leu Lys Ala Gln Asn Lys His Glu Leu 625 630 635 640 Ser TyrArg Lys Phe Arg Ala Lys Ile Cys Pro Gly Glu Asn Gln Ala 645 650 655 AlaGlu Asp Glu Leu Arg Lys Glu Ile Ser Lys Ser Met Phe Ala Glu 660 665 670Met Glu Ile Leu Gly Gln Phe Asn Leu Gly Phe Ile Val Thr Lys Leu 675 680685 Lys Glu Asp Leu Phe Leu Val Asp Gln His Ala Ala Asp Glu Lys Tyr 690695 700 Asn Phe Glu Met Leu Gln Gln His Thr Val Leu Gln Ala Gln Arg Leu705 710 715 720 Ile Thr Pro Gln Thr Leu Asn Leu Thr Ala Val Asn Glu AlaVal Leu 725 730 735 Ile Glu Asn Leu Glu Ile Phe Arg Lys Asn Gly Phe AspPhe Val Ile 740 745 750 Asp Glu Asp Ala Pro Val Thr Glu Arg Ala Lys LeuIle Ser Leu Pro 755 760 765 Thr Ser Lys Asn Trp Thr Phe Gly Pro Gln AspIle Asp Glu Leu Ile 770 775 780 Phe Met Leu Ser Asp Ser Pro Gly Val MetCys Arg Pro Ser Arg Val 785 790 795 800 Arg Gln Met Phe Ala Ser Arg AlaCys Arg Lys Ser Val Met Ile Gly 805 810 815 Thr Ala Leu Asn Ala Ser GluMet Lys Lys Leu Ile Thr His Met Gly 820 825 830 Glu Met Asp His Pro TrpAsn Cys Pro His Gly Arg Pro Thr Met Arg 835 840 845 His Val Ala Asn LeuAsp Val Ile Ser Gln Asn 850 855 5 2771 DNA Homo sapiens 5 cgaggcggatcgggtgttgc atccatggag cgagctgaga gctcgagtac agaacctgct 60 aaggccatcaaacctattga tcggaagtca gtccatcaga tttgctctgg gcaggtggta 120 ctgagtctaagcactgcggt aaaggagtta gtagaaaaca gtctggatgc tggtgccact 180 aatattgatctaaagcttaa ggactatgga gtggatctta ttgaagtttc agacaatgga 240 tgtggggtagaagaagaaaa cttcgaaggc ttaactctga aacatcacac atctaagatt 300 caagagtttgccgacctaac tcaggttgaa acttttggct ttcgggggga agctctgagc 360 tcactttgtgcactgagcga tgtcaccatt tctacctgcc acgcatcggc gaaggttgga 420 actcgactgatgtttgatca caatgggaaa attatccaga aaacccccta cccccgcccc 480 agagggaccacagtcagcgt gcagcagtta ttttccacac tacctgtgcg ccataaggaa 540 tttcaaaggaatattaagaa ggagtatgcc aaaatggtcc aggtcttaca tgcatactgt 600 atcatttcagcaggcatccg tgtaagttgc accaatcagc ttggacaagg aaaacgacag 660 cctgtggtatgcacaggtgg aagccccagc ataaaggaaa atatcggctc tgtgtttggg 720 cagaagcagttgcaaagcct cattcctttt gttcagctgc cccctagtga ctccgtgtgt 780 gaagagtacggtttgagctg ttcggatgct ctgcataatc ttttttacat ctcaggtttc 840 atttcacaatgcacgcatgg agttggaagg agttcaacag acagacagtt tttctttatc 900 aaccggcggccttgtgaccc agcaaaggtc tgcagactcg tgaatgaggt ctaccacatg 960 tataatcgacaccagtatcc atttgttgtt cttaacattt ctgttgattc agaatgcgtt 1020 gatatcaatgttactccaga taaaaggcaa attttgctac aagaggaaaa gcttttgttg 1080 gcagttttaaagacctcttt gataggaatg tttgatagtg atgtcaacaa gctaaatgtc 1140 agtcagcagccactgctgga tgttgaaggt aacttaataa aaatgcatgc agcggatttg 1200 gaaaagcccatggtagaaaa gcaggatcaa tccccttcat taaggactgg agaagaaaaa 1260 aaagacgtgtccatttccag actgcgagag gccttttctc ttcgtcacac aacagagaac 1320 aagcctcacagcccaaagac tccagaacca agaaggagcc ctctaggaca gaaaaggggt 1380 atgctgtcttctagcacttc aggtgccatc tctgacaaag gcgtcctgag acctcagaaa 1440 gaggcagtgagttccagtca cggacccagt gaccctacgg acagagcgga ggtggagaag 1500 gactcggggcacggcagcac ttccgtggat tctgaggggt tcagcatccc agacacgggc 1560 agtcactgcagcagcgagta tgcggccagc tccccagggg acaggggctc gcaggaacat 1620 gtggactctcaggagaaagc gcctgaaact gacgactctt tttcagatgt ggactgccat 1680 tcaaaccaggaagataccgg atgtaaattt cgagttttgc ctcagccaac taatctcgca 1740 accccaaacacaaagcgttt taaaaaagaa gaaattcttt ccagttctga catttgtcaa 1800 aagttagtaaatactcagga catgtcagcc tctcaggttg atgtagctgt gaaaattaat 1860 aagaaagttgtgcccctgga cttttctatg agttctttag ctaaacgaat aaagcagtta 1920 catcatgaagcacagcaaag tgaaggggaa cagaattaca ggaagtttag ggcaaagatt 1980 tgtcctggagaaaatcaagc agccgaagat gaactaagaa aagagataag taaaacgatg 2040 tttgcagaaatggaaatcat tggtcagttt aacctgggat ttataataac caaactgaat 2100 gaggatatcttcatagtgga ccagcatgcc acggacgaga agtataactt cgagatgctg 2160 cagcagcacaccgtgctcca ggggcagagg ctcatagcac ctcagactct caacttaact 2220 gctgttaatgaagctgttct gatagaaaat ctggaaatat ttagaaagaa tggctttgat 2280 tttgttatcgatgaaaatgc tccagtcact gaaagggcta aactgatttc cttgccaact 2340 agtaaaaactggaccttcgg accccaggac gtcgatgaac tgatcttcat gctgagcgac 2400 agccctggggtcatgtgccg gccttcccga gtcaagcaga tgtttgcctc cagagcctgc 2460 cggaagtcggtgatgattgg gactgctctt aacacaagcg agatgaagaa actgatcacc 2520 cacatgggggagatggacca cccctggaac tgtccccatg gaaggccaac catgagacac 2580 atcgccaacctgggtgtcat ttctcagaac tgaccgtagt cactgtatgg aataattggt 2640 tttatcgcagatttttatgt tttgaaagac agagtcttca ctaacctttt ttgttttaaa 2700 atgaaacctgctacttaaaa aaaatacaca tcacacccat ttaaaagtga tcttgagaac 2760 cttttcaaac c2771 6 932 PRT Homo sapiens 6 Met Lys Gln Leu Pro Ala Ala Thr Val ArgLeu Leu Ser Ser Ser Gln 1 5 10 15 Ile Ile Thr Ser Val Val Ser Val ValLys Glu Leu Ile Glu Asn Ser 20 25 30 Leu Asp Ala Gly Ala Thr Ser Val AspVal Lys Leu Glu Asn Tyr Gly 35 40 45 Phe Asp Lys Ile Glu Val Arg Asp AsnGly Glu Gly Ile Lys Ala Val 50 55 60 Asp Ala Pro Val Met Ala Met Lys TyrTyr Thr Ser Lys Ile Asn Ser 65 70 75 80 His Glu Asp Leu Glu Asn Leu ThrThr Tyr Gly Phe Arg Gly Glu Ala 85 90 95 Leu Gly Ser Ile Cys Cys Ile AlaGlu Val Leu Ile Thr Thr Arg Thr 100 105 110 Ala Ala Asp Asn Phe Ser ThrGln Tyr Val Leu Asp Gly Ser Gly His 115 120 125 Ile Leu Ser Gln Lys ProSer His Leu Gly Gln Gly Thr Thr Val Thr 130 135 140 Ala Leu Arg Leu PheLys Asn Leu Pro Val Arg Lys Gln Phe Tyr Ser 145 150 155 160 Thr Ala LysLys Cys Lys Asp Glu Ile Lys Lys Ile Gln Asp Leu Leu 165 170 175 Met SerPhe Gly Ile Leu Lys Pro Asp Leu Arg Ile Val Phe Val His 180 185 190 AsnLys Ala Val Ile Trp Gln Lys Ser Arg Val Ser Asp His Lys Met 195 200 205Ala Leu Met Ser Val Leu Gly Thr Ala Val Met Asn Asn Met Glu Ser 210 215220 Phe Gln Tyr His Ser Glu Glu Ser Gln Ile Tyr Leu Ser Gly Phe Leu 225230 235 240 Pro Lys Cys Asp Ala Asp His Ser Phe Thr Ser Leu Ser Thr ProGlu 245 250 255 Arg Ser Phe Ile Phe Ile Asn Ser Arg Pro Val His Gln LysAsp Ile 260 265 270 Leu Lys Leu Ile Arg His His Tyr Asn Leu Lys Cys LeuLys Glu Ser 275 280 285 Thr Arg Leu Tyr Pro Val Phe Phe Leu Lys Ile AspVal Pro Thr Ala 290 295 300 Asp Val Asp Val Asn Leu Thr Pro Asp Lys SerGln Val Leu Leu Gln 305 310 315 320 Asn Lys Glu Ser Val Leu Ile Ala LeuGlu Asn Leu Met Thr Thr Cys 325 330 335 Tyr Gly Pro Leu Pro Ser Thr AsnSer Tyr Glu Asn Asn Lys Thr Asp 340 345 350 Val Ser Ala Ala Asp Ile ValLeu Ser Lys Thr Ala Glu Thr Asp Val 355 360 365 Leu Phe Asn Lys Val GluSer Ser Gly Lys Asn Tyr Ser Asn Val Asp 370 375 380 Thr Ser Val Ile ProPhe Gln Asn Asp Met His Asn Asp Glu Ser Gly 385 390 395 400 Lys Asn ThrAsp Asp Cys Leu Asn His Gln Ile Ser Ile Gly Asp Phe 405 410 415 Gly TyrGly His Cys Ser Ser Glu Ile Ser Asn Ile Asp Lys Asn Thr 420 425 430 LysAsn Ala Phe Gln Asp Ile Ser Met Ser Asn Val Ser Trp Glu Asn 435 440 445Ser Gln Thr Glu Tyr Ser Lys Thr Cys Phe Ile Ser Ser Val Lys His 450 455460 Thr Gln Ser Glu Asn Gly Asn Lys Asp His Ile Asp Glu Ser Gly Glu 465470 475 480 Asn Glu Glu Glu Ala Gly Leu Glu Asn Ser Ser Glu Ile Ser AlaAsp 485 490 495 Glu Trp Ser Arg Gly Asn Ile Leu Lys Asn Ser Val Gly GluAsn Ile 500 505 510 Glu Pro Val Lys Ile Leu Val Pro Glu Lys Ser Leu ProCys Lys Val 515 520 525 Ser Asn Asn Asn Tyr Pro Ile Pro Glu Gln Met AsnLeu Asn Glu Asp 530 535 540 Ser Cys Asn Lys Lys Ser Asn Val Ile Asp AsnLys Ser Gly Lys Val 545 550 555 560 Thr Ala Tyr Asp Leu Leu Ser Asn ArgVal Ile Lys Lys Pro Met Ser 565 570 575 Ala Ser Ala Leu Phe Val Gln AspHis Arg Pro Gln Phe Leu Ile Glu 580 585 590 Asn Pro Lys Thr Ser Leu GluAsp Ala Thr Leu Gln Ile Glu Glu Leu 595 600 605 Trp Lys Thr Leu Ser GluGlu Glu Lys Leu Lys Tyr Glu Glu Lys Ala 610 615 620 Thr Lys Asp Leu GluArg Tyr Asn Ser Gln Met Lys Arg Ala Ile Glu 625 630 635 640 Gln Glu SerGln Met Ser Leu Lys Asp Gly Arg Lys Lys Ile Lys Pro 645 650 655 Thr SerAla Trp Asn Leu Ala Gln Lys His Lys Leu Lys Thr Ser Leu 660 665 670 SerAsn Gln Pro Lys Leu Asp Glu Leu Leu Gln Ser Gln Ile Glu Lys 675 680 685Arg Arg Ser Gln Asn Ile Lys Met Val Gln Ile Pro Phe Ser Met Lys 690 695700 Asn Leu Lys Ile Asn Phe Lys Lys Gln Asn Lys Val Asp Leu Glu Glu 705710 715 720 Lys Asp Glu Pro Cys Leu Ile His Asn Leu Arg Phe Pro Asp AlaTrp 725 730 735 Leu Met Thr Ser Lys Thr Glu Val Met Leu Leu Asn Pro TyrArg Val 740 745 750 Glu Glu Ala Leu Leu Phe Lys Arg Leu Leu Glu Asn HisLys Leu Pro 755 760 765 Ala Glu Pro Leu Glu Lys Pro Ile Met Leu Thr GluSer Leu Phe Asn 770 775 780 Gly Ser His Tyr Leu Asp Val Leu Tyr Lys MetThr Ala Asp Asp Gln 785 790 795 800 Arg Tyr Ser Gly Ser Thr Tyr Leu SerAsp Pro Arg Leu Thr Ala Asn 805 810 815 Gly Phe Lys Ile Lys Leu Ile ProGly Val Ser Ile Thr Glu Asn Tyr 820 825 830 Leu Glu Ile Glu Gly Met AlaAsn Cys Leu Pro Phe Tyr Gly Val Ala 835 840 845 Asp Leu Lys Glu Ile LeuAsn Ala Ile Leu Asn Arg Asn Ala Lys Glu 850 855 860 Val Tyr Glu Cys ArgPro Arg Lys Val Ile Ser Tyr Leu Glu Gly Glu 865 870 875 880 Ala Val ArgLeu Ser Arg Gln Leu Pro Met Tyr Leu Ser Lys Glu Asp 885 890 895 Ile GlnAsp Ile Ile Tyr Arg Met Lys His Gln Phe Gly Asn Glu Ile 900 905 910 LysGlu Cys Val His Gly Arg Pro Phe Phe His His Leu Thr Tyr Leu 915 920 925Pro Glu Thr Thr 930 7 3063 DNA Homo sapiens 7 ggcacgagtg gctgcttgcggctagtggat ggtaattgcc tgcctcgcgc tagcagcaag 60 ctgctctgtt aaaagcgaaaatgaaacaat tgcctgcggc aacagttcga ctcctttcaa 120 gttctcagat catcacttcggtggtcagtg ttgtaaaaga gcttattgaa aactccttgg 180 atgctggtgc cacaagcgtagatgttaaac tggagaacta tggatttgat aaaattgagg 240 tgcgagataa cggggagggtatcaaggctg ttgatgcacc tgtaatggca atgaagtact 300 acacctcaaa aataaatagtcatgaagatc ttgaaaattt gacaacttac ggttttcgtg 360 gagaagcctt ggggtcaatttgttgtatag ctgaggtttt aattacaaca agaacggctg 420 ctgataattt tagcacccagtatgttttag atggcagtgg ccacatactt tctcagaaac 480 cttcacatct tggtcaaggtacaactgtaa ctgctttaag attatttaag aatctacctg 540 taagaaagca gttttactcaactgcaaaaa aatgtaaaga tgaaataaaa aagatccaag 600 atctcctcat gagctttggtatccttaaac ctgacttaag gattgtcttt gtacataaca 660 aggcagttat ttggcagaaaagcagagtat cagatcacaa gatggctctc atgtcagttc 720 tggggactgc tgttatgaacaatatggaat cctttcagta ccactctgaa gaatctcaga 780 tttatctcag tggatttcttccaaagtgtg atgcagacca ctctttcact agtctttcaa 840 caccagaaag aagtttcatcttcataaaca gtcgaccagt acatcaaaaa gatatcttaa 900 agttaatccg acatcattacaatctgaaat gcctaaagga atctactcgt ttgtatcctg 960 ttttctttct gaaaatcgatgttcctacag ctgatgttga tgtaaattta acaccagata 1020 aaagccaagt attattacaaaataaggaat ctgttttaat tgctcttgaa aatctgatga 1080 cgacttgtta tggaccattacctagtacaa attcttatga aaataataaa acagatgttt 1140 ccgcagctga catcgttcttagtaaaacag cagaaacaga tgtgcttttt aataaagtgg 1200 aatcatctgg aaagaattattcaaatgttg atacttcagt cattccattc caaaatgata 1260 tgcataatga tgaatctggaaaaaacactg atgattgttt aaatcaccag ataagtattg 1320 gtgactttgg ttatggtcattgtagtagtg aaatttctaa cattgataaa aacactaaga 1380 atgcatttca ggacatttcaatgagtaatg tatcatggga gaactctcag acggaatata 1440 gtaaaacttg ttttataagttccgttaagc acacccagtc agaaaatggc aataaagacc 1500 atatagatga gagtggggaaaatgaggaag aagcaggtct tgaaaactct tcggaaattt 1560 ctgcagatga gtggagcaggggaaatatac ttaaaaattc agtgggagag aatattgaac 1620 ctgtgaaaat tttagtgcctgaaaaaagtt taccatgtaa agtaagtaat aataattatc 1680 caatccctga acaaatgaatcttaatgaag attcatgtaa caaaaaatca aatgtaatag 1740 ataataaatc tggaaaagttacagcttatg atttacttag caatcgagta atcaagaaac 1800 ccatgtcagc aagtgctctttttgttcaag atcatcgtcc tcagtttctc atagaaaatc 1860 ctaagactag tttagaggatgcaacactac aaattgaaga actgtggaag acattgagtg 1920 aagaggaaaa actgaaatatgaagagaagg ctactaaaga cttggaacga tacaatagtc 1980 aaatgaagag agccattgaacaggagtcac aaatgtcact aaaagatggc agaaaaaaga 2040 taaaacccac cagcgcatggaatttggccc agaagcacaa gttaaaaacc tcattatcta 2100 atcaaccaaa acttgatgaactccttcagt cccaaattga aaaaagaagg agtcaaaata 2160 ttaaaatggt acagatccccttttctatga aaaacttaaa aataaatttt aagaaacaaa 2220 acaaagttga cttagaagagaaggatgaac cttgcttgat ccacaatctc aggtttcctg 2280 atgcatggct aatgacatccaaaacagagg taatgttatt aaatccatat agagtagaag 2340 aagccctgct atttaaaagacttcttgaga atcataaact tcctgcagag ccactggaaa 2400 agccaattat gttaacagagagtcttttta atggatctca ttatttagac gttttatata 2460 aaatgacagc agatgaccaaagatacagtg gatcaactta cctgtctgat cctcgtctta 2520 cagcgaatgg tttcaagataaaattgatac caggagtttc aattactgaa aattacttgg 2580 aaatagaagg aatggctaattgtctcccat tctatggagt agcagattta aaagaaattc 2640 ttaatgctat attaaacagaaatgcaaagg aagtttatga atgtagacct cgcaaagtga 2700 taagttattt agagggagaagcagtgcgtc tatccagaca attacccatg tacttatcaa 2760 aagaggacat ccaagacattatctacagaa tgaagcacca gtttggaaat gaaattaaag 2820 agtgtgttca tggtcgcccattttttcatc atttaaccta tcttccagaa actacatgat 2880 taaatatgtt taagaagattagttaccatt gaaattggtt ctgtcataaa acagcatgag 2940 tctggtttta aattatctttgtattatgtg tcacatggtt attttttaaa tgaggattca 3000 ctgacttgtt tttatattgaaaaaagttcc acgtattgta gaaaacgtaa ataaactaat 3060 aac 3063 8 932 PRT Homosapiens 8 Met Lys Gln Leu Pro Ala Ala Thr Val Arg Leu Leu Ser Ser SerGln 1 5 10 15 Ile Ile Thr Ser Val Val Ser Val Val Lys Glu Leu Ile GluAsn Ser 20 25 30 Leu Asp Ala Gly Ala Thr Ser Val Asp Val Lys Leu Glu AsnTyr Gly 35 40 45 Phe Asp Lys Ile Glu Val Arg Asp Asn Gly Glu Gly Ile LysAla Val 50 55 60 Asp Ala Pro Val Met Ala Met Lys Tyr Tyr Thr Ser Lys IleAsn Ser 65 70 75 80 His Glu Asp Leu Glu Asn Leu Thr Thr Tyr Gly Phe ArgGly Glu Ala 85 90 95 Leu Gly Ser Ile Cys Cys Ile Ala Glu Val Leu Ile ThrThr Arg Thr 100 105 110 Ala Ala Asp Asn Phe Ser Thr Gln Tyr Val Leu AspGly Ser Gly His 115 120 125 Ile Leu Ser Gln Lys Pro Ser His Leu Gly GlnGly Thr Thr Val Thr 130 135 140 Ala Leu Arg Leu Phe Lys Asn Leu Pro ValArg Lys Gln Phe Tyr Ser 145 150 155 160 Thr Ala Lys Lys Cys Lys Asp GluIle Lys Lys Ile Gln Asp Leu Leu 165 170 175 Met Ser Phe Gly Ile Leu LysPro Asp Leu Arg Ile Val Phe Val His 180 185 190 Asn Lys Ala Val Ile TrpGln Lys Ser Arg Val Ser Asp His Lys Met 195 200 205 Ala Leu Met Ser ValLeu Gly Thr Ala Val Met Asn Asn Met Glu Ser 210 215 220 Phe Gln Tyr HisSer Glu Glu Ser Gln Ile Tyr Leu Ser Gly Phe Leu 225 230 235 240 Pro LysCys Asp Ala Asp His Ser Phe Thr Ser Leu Ser Thr Pro Glu 245 250 255 ArgSer Phe Ile Phe Ile Asn Ser Arg Pro Val His Gln Lys Asp Ile 260 265 270Leu Lys Leu Ile Arg His His Tyr Asn Leu Lys Cys Leu Lys Glu Ser 275 280285 Thr Arg Leu Tyr Pro Val Phe Phe Leu Lys Ile Asp Val Pro Thr Ala 290295 300 Asp Val Asp Val Asn Leu Thr Pro Asp Lys Ser Gln Val Leu Leu Gln305 310 315 320 Asn Lys Glu Ser Val Leu Ile Ala Leu Glu Asn Leu Met ThrThr Cys 325 330 335 Tyr Gly Pro Leu Pro Ser Thr Asn Ser Tyr Glu Asn AsnLys Thr Asp 340 345 350 Val Ser Ala Ala Asp Ile Val Leu Ser Lys Thr AlaGlu Thr Asp Val 355 360 365 Leu Phe Asn Lys Val Glu Ser Ser Gly Lys AsnTyr Ser Asn Val Asp 370 375 380 Thr Ser Val Ile Pro Phe Gln Asn Asp MetHis Asn Asp Glu Ser Gly 385 390 395 400 Lys Asn Thr Asp Asp Cys Leu AsnHis Gln Ile Ser Ile Gly Asp Phe 405 410 415 Gly Tyr Gly His Cys Ser SerGlu Ile Ser Asn Ile Asp Lys Asn Thr 420 425 430 Lys Asn Ala Phe Gln AspIle Ser Met Ser Asn Val Ser Trp Glu Asn 435 440 445 Ser Gln Thr Glu TyrSer Lys Thr Cys Phe Ile Ser Ser Val Lys His 450 455 460 Thr Gln Ser GluAsn Gly Asn Lys Asp His Ile Asp Glu Ser Gly Glu 465 470 475 480 Asn GluGlu Glu Ala Gly Leu Glu Asn Ser Ser Glu Ile Ser Ala Asp 485 490 495 GluTrp Ser Arg Gly Asn Ile Leu Lys Asn Ser Val Gly Glu Asn Ile 500 505 510Glu Pro Val Lys Ile Leu Val Pro Glu Lys Ser Leu Pro Cys Lys Val 515 520525 Ser Asn Asn Asn Tyr Pro Ile Pro Glu Gln Met Asn Leu Asn Glu Asp 530535 540 Ser Cys Asn Lys Lys Ser Asn Val Ile Asp Asn Lys Ser Gly Lys Val545 550 555 560 Thr Ala Tyr Asp Leu Leu Ser Asn Arg Val Ile Lys Lys ProMet Ser 565 570 575 Ala Ser Ala Leu Phe Val Gln Asp His Arg Pro Gln PheLeu Ile Glu 580 585 590 Asn Pro Lys Thr Ser Leu Glu Asp Ala Thr Leu GlnIle Glu Glu Leu 595 600 605 Trp Lys Thr Leu Ser Glu Glu Glu Lys Leu LysTyr Glu Glu Lys Ala 610 615 620 Thr Lys Asp Leu Glu Arg Tyr Asn Ser GlnMet Lys Arg Ala Ile Glu 625 630 635 640 Gln Glu Ser Gln Met Ser Leu LysAsp Gly Arg Lys Lys Ile Lys Pro 645 650 655 Thr Ser Ala Trp Asn Leu AlaGln Lys His Lys Leu Lys Thr Ser Leu 660 665 670 Ser Asn Gln Pro Lys LeuAsp Glu Leu Leu Gln Ser Gln Ile Glu Lys 675 680 685 Arg Arg Ser Gln AsnIle Lys Met Val Gln Ile Pro Phe Ser Met Lys 690 695 700 Asn Leu Lys IleAsn Phe Lys Lys Gln Asn Lys Val Asp Leu Glu Glu 705 710 715 720 Lys AspGlu Pro Cys Leu Ile His Asn Leu Arg Phe Pro Asp Ala Trp 725 730 735 LeuMet Thr Ser Lys Thr Glu Val Met Leu Leu Asn Pro Tyr Arg Val 740 745 750Glu Glu Ala Leu Leu Phe Lys Arg Leu Leu Glu Asn His Lys Leu Pro 755 760765 Ala Glu Pro Leu Glu Lys Pro Ile Met Leu Thr Glu Ser Leu Phe Asn 770775 780 Gly Ser His Tyr Leu Asp Val Leu Tyr Lys Met Thr Ala Asp Asp Gln785 790 795 800 Arg Tyr Ser Gly Ser Thr Tyr Leu Ser Asp Pro Arg Leu ThrAla Asn 805 810 815 Gly Phe Lys Ile Lys Leu Ile Pro Gly Val Ser Ile ThrGlu Asn Tyr 820 825 830 Leu Glu Ile Glu Gly Met Ala Asn Cys Leu Pro PheTyr Gly Val Ala 835 840 845 Asp Leu Lys Glu Ile Leu Asn Ala Ile Leu AsnArg Asn Ala Lys Glu 850 855 860 Val Tyr Glu Cys Arg Pro Arg Lys Val IleSer Tyr Leu Glu Gly Glu 865 870 875 880 Ala Val Arg Leu Ser Arg Gln LeuPro Met Tyr Leu Ser Lys Glu Asp 885 890 895 Ile Gln Asp Ile Ile Tyr ArgMet Lys His Gln Phe Gly Asn Glu Ile 900 905 910 Lys Glu Cys Val His GlyArg Pro Phe Phe His His Leu Thr Tyr Leu 915 920 925 Pro Glu Thr Thr 9309 3145 DNA Homo sapiens 9 ggcgggaaac agcttagtgg gtgtggggtc gcgcattttcttcaaccagg aggtgaggag 60 gtttcgacat ggcggtgcag ccgaaggaga cgctgcagttggagagcgcg gccgaggtcg 120 gcttcgtgcg cttctttcag ggcatgccgg agaagccgaccaccacagtg cgccttttcg 180 accggggcga cttctatacg gcgcacggcg aggacgcgctgctggccgcc cgggaggtgt 240 tcaagaccca gggggtgatc aagtacatgg ggccggcaggagcaaagaat ctgcagagtg 300 ttgtgcttag taaaatgaat tttgaatctt ttgtaaaagatcttcttctg gttcgtcagt 360 atagagttga agtttataag aatagagctg gaaataaggcatccaaggag aatgattggt 420 atttggcata taaggcttct cctggcaatc tctctcagtttgaagacatt ctctttggta 480 acaatgatat gtcagcttcc attggtgttg tgggtgttaaaatgtccgca gttgatggcc 540 agagacaggt tggagttggg tatgtggatt ccatacagaggaaactagga ctgtgtgaat 600 tccctgataa tgatcagttc tccaatcttg aggctctcctcatccagatt ggaccaaagg 660 aatgtgtttt acccggagga gagactgctg gagacatggggaaactgaga cagataattc 720 aaagaggagg aattctgatc acagaaagaa aaaaagctgacttttccaca aaagacattt 780 atcaggacct caaccggttg ttgaaaggca aaaagggagagcagatgaat agtgctgtat 840 tgccagaaat ggagaatcag gttgcagttt catcactgtctgcggtaatc aagtttttag 900 aactcttatc agatgattcc aactttggac agtttgaactgactactttt gacttcagcc 960 agtatatgaa attggatatt gcagcagtca gagcccttaacctttttcag ggttctgttg 1020 aagataccac tggctctcag tctctggctg ccttgctgaataagtgtaaa acccctcaag 1080 gacaaagact tgttaaccag tggattaagc agcctctcatggataagaac agaatagagg 1140 agagattgaa tttagtggaa gcttttgtag aagatgcagaattgaggcag actttacaag 1200 aagatttact tcgtcgattc ccagatctta accgacttgccaagaagttt caaagacaag 1260 cagcaaactt acaagattgt taccgactct atcagggtataaatcaacta cctaatgtta 1320 tacaggctct ggaaaaacat gaaggaaaac accagaaattattgttggca gtttttgtga 1380 ctcctcttac tgatcttcgt tctgacttct ccaagtttcaggaaatgata gaaacaactt 1440 tagatatgga tcaggtggaa aaccatgaat tccttgtaaaaccttcattt gatcctaatc 1500 tcagtgaatt aagagaaata atgaatgact tggaaaagaagatgcagtca acattaataa 1560 gtgcagccag agatcttggc ttggaccctg gcaaacagattaaactggat tccagtgcac 1620 agtttggata ttactttcgt gtaacctgta aggaagaaaaagtccttcgt aacaataaaa 1680 actttagtac tgtagatatc cagaagaatg gtgttaaatttaccaacagc aaattgactt 1740 ctttaaatga agagtatacc aaaaataaaa cagaatatgaagaagcccag gatgccattg 1800 ttaaagaaat tgtcaatatt tcttcaggct atgtagaaccaatgcagaca ctcaatgatg 1860 tgttagctca gctagatgct gttgtcagct ttgctcacgtgtcaaatgga gcacctgttc 1920 catatgtacg accagccatt ttggagaaag gacaaggaagaattatatta aaagcatcca 1980 ggcatgcttg tgttgaagtt caagatgaaa ttgcatttattcctaatgac gtatactttg 2040 aaaaagataa acagatgttc cacatcatta ctggccccaatatgggaggt aaatcaacat 2100 atattcgaca aactggggtg atagtactca tggcccaaattgggtgtttt gtgccatgtg 2160 agtcagcaga agtgtccatt gtggactgca tcttagcccgagtaggggct ggtgacagtc 2220 aattgaaagg agtctccacg ttcatggctg aaatgttggaaactgcttct atcctcaggt 2280 ctgcaaccaa agattcatta ataatcatag atgaattgggaagaggaact tctacctacg 2340 atggatttgg gttagcatgg gctatatcag aatacattgcaacaaagatt ggtgcttttt 2400 gcatgtttgc aacccatttt catgaactta ctgccttggccaatcagata ccaactgtta 2460 ataatctaca tgtcacagca ctcaccactg aagagaccttaactatgctt tatcaggtga 2520 agaaaggtgt ctgtgatcaa agttttggga ttcatgttgcagagcttgct aatttcccta 2580 agcatgtaat agagtgtgct aaacagaaag ccctggaacttgaggagttt cagtatattg 2640 gagaatcgca aggatatgat atcatggaac cagcagcaaagaagtgctat ctggaaagag 2700 agcaaggtga aaaaattatt caggagttcc tgtccaaggtgaaacaaatg ccctttactg 2760 aaatgtcaga agaaaacatc acaataaagt taaaacagctaaaagctgaa gtaatagcaa 2820 agaataatag ctttgtaaat gaaatcattt cacgaataaaagttactacg tgaaaaatcc 2880 cagtaatgga atgaaggtaa tattgataag ctattgtctgtaatagtttt atattgtttt 2940 atattaaccc tttttccata gtgttaactg tcagtgcccatgggctatca acttaataag 3000 atatttagta atattttact ttgaggacat tttcaaagatttttattttg aaaaatgaga 3060 gctgtaactg aggactgttt gcaattgaca taggcaataataagtgatgt gctgaatttt 3120 ataaataaaa tcatgtagtt tgtgg 3145 10 934 PRTHomo sapiens 10 Met Ala Val Gln Pro Lys Glu Thr Leu Gln Leu Glu Ser AlaAla Glu 1 5 10 15 Val Gly Phe Val Arg Phe Phe Gln Gly Met Pro Glu LysPro Thr Thr 20 25 30 Thr Val Arg Leu Phe Asp Arg Gly Asp Phe Tyr Thr AlaHis Gly Glu 35 40 45 Asp Ala Leu Leu Ala Ala Arg Glu Val Phe Lys Thr GlnGly Val Ile 50 55 60 Lys Tyr Met Gly Pro Ala Gly Ala Lys Asn Leu Gln SerVal Val Leu 65 70 75 80 Ser Lys Met Asn Phe Glu Ser Phe Val Lys Asp LeuLeu Leu Val Arg 85 90 95 Gln Tyr Arg Val Glu Val Tyr Lys Asn Arg Ala GlyAsn Lys Ala Ser 100 105 110 Lys Glu Asn Asp Trp Tyr Leu Ala Tyr Lys AlaSer Pro Gly Asn Leu 115 120 125 Ser Gln Phe Glu Asp Ile Leu Phe Gly AsnAsn Asp Met Ser Ala Ser 130 135 140 Ile Gly Val Val Gly Val Lys Met SerAla Val Asp Gly Gln Arg Gln 145 150 155 160 Val Gly Val Gly Tyr Val AspSer Ile Gln Arg Lys Leu Gly Leu Cys 165 170 175 Glu Phe Pro Asp Asn AspGln Phe Ser Asn Leu Glu Ala Leu Leu Ile 180 185 190 Gln Ile Gly Pro LysGlu Cys Val Leu Pro Gly Gly Glu Thr Ala Gly 195 200 205 Asp Met Gly LysLeu Arg Gln Ile Ile Gln Arg Gly Gly Ile Leu Ile 210 215 220 Thr Glu ArgLys Lys Ala Asp Phe Ser Thr Lys Asp Ile Tyr Gln Asp 225 230 235 240 LeuAsn Arg Leu Leu Lys Gly Lys Lys Gly Glu Gln Met Asn Ser Ala 245 250 255Val Leu Pro Glu Met Glu Asn Gln Val Ala Val Ser Ser Leu Ser Ala 260 265270 Val Ile Lys Phe Leu Glu Leu Leu Ser Asp Asp Ser Asn Phe Gly Gln 275280 285 Phe Glu Leu Thr Thr Phe Asp Phe Ser Gln Tyr Met Lys Leu Asp Ile290 295 300 Ala Ala Val Arg Ala Leu Asn Leu Phe Gln Gly Ser Val Glu AspThr 305 310 315 320 Thr Gly Ser Gln Ser Leu Ala Ala Leu Leu Asn Lys CysLys Thr Pro 325 330 335 Gln Gly Gln Arg Leu Val Asn Gln Trp Ile Lys GlnPro Leu Met Asp 340 345 350 Lys Asn Arg Ile Glu Glu Arg Leu Asn Leu ValGlu Ala Phe Val Glu 355 360 365 Asp Ala Glu Leu Arg Gln Thr Leu Gln GluAsp Leu Leu Arg Arg Phe 370 375 380 Pro Asp Leu Asn Arg Leu Ala Lys LysPhe Gln Arg Gln Ala Ala Asn 385 390 395 400 Leu Gln Asp Cys Tyr Arg LeuTyr Gln Gly Ile Asn Gln Leu Pro Asn 405 410 415 Val Ile Gln Ala Leu GluLys His Glu Gly Lys His Gln Lys Leu Leu 420 425 430 Leu Ala Val Phe ValThr Pro Leu Thr Asp Leu Arg Ser Asp Phe Ser 435 440 445 Lys Phe Gln GluMet Ile Glu Thr Thr Leu Asp Met Asp Gln Val Glu 450 455 460 Asn His GluPhe Leu Val Lys Pro Ser Phe Asp Pro Asn Leu Ser Glu 465 470 475 480 LeuArg Glu Ile Met Asn Asp Leu Glu Lys Lys Met Gln Ser Thr Leu 485 490 495Ile Ser Ala Ala Arg Asp Leu Gly Leu Asp Pro Gly Lys Gln Ile Lys 500 505510 Leu Asp Ser Ser Ala Gln Phe Gly Tyr Tyr Phe Arg Val Thr Cys Lys 515520 525 Glu Glu Lys Val Leu Arg Asn Asn Lys Asn Phe Ser Thr Val Asp Ile530 535 540 Gln Lys Asn Gly Val Lys Phe Thr Asn Ser Lys Leu Thr Ser LeuAsn 545 550 555 560 Glu Glu Tyr Thr Lys Asn Lys Thr Glu Tyr Glu Glu AlaGln Asp Ala 565 570 575 Ile Val Lys Glu Ile Val Asn Ile Ser Ser Gly TyrVal Glu Pro Met 580 585 590 Gln Thr Leu Asn Asp Val Leu Ala Gln Leu AspAla Val Val Ser Phe 595 600 605 Ala His Val Ser Asn Gly Ala Pro Val ProTyr Val Arg Pro Ala Ile 610 615 620 Leu Glu Lys Gly Gln Gly Arg Ile IleLeu Lys Ala Ser Arg His Ala 625 630 635 640 Cys Val Glu Val Gln Asp GluIle Ala Phe Ile Pro Asn Asp Val Tyr 645 650 655 Phe Glu Lys Asp Lys GlnMet Phe His Ile Ile Thr Gly Pro Asn Met 660 665 670 Gly Gly Lys Ser ThrTyr Ile Arg Gln Thr Gly Val Ile Val Leu Met 675 680 685 Ala Gln Ile GlyCys Phe Val Pro Cys Glu Ser Ala Glu Val Ser Ile 690 695 700 Val Asp CysIle Leu Ala Arg Val Gly Ala Gly Asp Ser Gln Leu Lys 705 710 715 720 GlyVal Ser Thr Phe Met Ala Glu Met Leu Glu Thr Ala Ser Ile Leu 725 730 735Arg Ser Ala Thr Lys Asp Ser Leu Ile Ile Ile Asp Glu Leu Gly Arg 740 745750 Gly Thr Ser Thr Tyr Asp Gly Phe Gly Leu Ala Trp Ala Ile Ser Glu 755760 765 Tyr Ile Ala Thr Lys Ile Gly Ala Phe Cys Met Phe Ala Thr His Phe770 775 780 His Glu Leu Thr Ala Leu Ala Asn Gln Ile Pro Thr Val Asn AsnLeu 785 790 795 800 His Val Thr Ala Leu Thr Thr Glu Glu Thr Leu Thr MetLeu Tyr Gln 805 810 815 Val Lys Lys Gly Val Cys Asp Gln Ser Phe Gly IleHis Val Ala Glu 820 825 830 Leu Ala Asn Phe Pro Lys His Val Ile Glu CysAla Lys Gln Lys Ala 835 840 845 Leu Glu Leu Glu Glu Phe Gln Tyr Ile GlyGlu Ser Gln Gly Tyr Asp 850 855 860 Ile Met Glu Pro Ala Ala Lys Lys CysTyr Leu Glu Arg Glu Gln Gly 865 870 875 880 Glu Lys Ile Ile Gln Glu PheLeu Ser Lys Val Lys Gln Met Pro Phe 885 890 895 Thr Glu Met Ser Glu GluAsn Ile Thr Ile Lys Leu Lys Gln Leu Lys 900 905 910 Ala Glu Val Ile AlaLys Asn Asn Ser Phe Val Asn Glu Ile Ile Ser 915 920 925 Arg Ile Lys ValThr Thr 930 11 2484 DNA Homo sapiens 11 cttggctctt ctggcgccaa aatgtcgttcgtggcagggg ttattcggcg gctggacgag 60 acagtggtga accgcatcgc ggcgggggaagttatccagc ggccagctaa tgctatcaaa 120 gagatgattg agaactgttt agatgcaaaatccacaagta ttcaagtgat tgttaaagag 180 ggaggcctga agttgattca gatccaagacaatggcaccg ggatcaggaa agaagatctg 240 gatattgtat gtgaaaggtt cactactagtaaactgcagt cctttgagga tttagccagt 300 atttctacct atggctttcg aggtgaggctttggccagca taagccatgt ggctcatgtt 360 actattacaa cgaaaacagc tgatggaaagtgtgcataca gagcaagtta ctcagatgga 420 aaactgaaag cccctcctaa accatgtgctggcaatcaag ggacccagat cacggtggag 480 gacctttttt acaacatagc cacgaggagaaaagctttaa aaaatccaag tgaagaatat 540 gggaaaattt tggaagttgt tggcaggtattcagtacaca atgcaggcat tagtttctca 600 gttaaaaaac aaggagagac agtagctgatgttaggacac tacccaatgc ctcaaccgtg 660 gacaatattc gctccatctt tggaaatgctgttagtcgag aactgataga aattggatgt 720 gaggataaaa ccctagcctt caaaatgaatggttacatat ccaatgcaaa ctactcagtg 780 aagaagtgca tcttcttact cttcatcaaccatcgtctgg tagaatcaac ttccttgaga 840 aaagccatag aaacagtgta tgcagcctatttgcccaaaa acacacaccc attcctgtac 900 ctcagtttag aaatcagtcc ccagaatgtggatgttaatg tgcaccccac aaagcatgaa 960 gttcacttcc tgcacgagga gagcatcctggagcgggtgc agcagcacat cgagagcaag 1020 ctcctgggct ccaattcctc caggatgtacttcacccaga ctttgctacc aggacttgct 1080 ggcccctctg gggagatggt taaatccacaacaagtctga cctcgtcttc tacttctgga 1140 agtagtgata aggtctatgc ccaccagatggttcgtacag attcccggga acagaagctt 1200 gatgcatttc tgcagcctct gagcaaacccctgtccagtc agccccaggc cattgtcaca 1260 gaggataaga cagatatttc tagtggcagggctaggcagc aagatgagga gatgcttgaa 1320 ctcccagccc ctgctgaagt ggctgccaaaaatcagagct tggaggggga tacaacaaag 1380 gggacttcag aaatgtcaga gaagagaggacctacttcca gcaaccccag aaagagacat 1440 cgggaagatt ctgatgtgga aatggtggaagatgattccc gaaaggaaat gactgcagct 1500 tgtacccccc ggagaaggat cattaacctcactagtgttt tgagtctcca ggaagaaatt 1560 aatgagcagg gacatgaggt tctccgggagatgttgcata accactcctt cgtgggctgt 1620 gtgaatcctc agtgggcctt ggcacagcatcaaaccaagt tataccttct caacaccacc 1680 aagcttagtg aagaactgtt ctaccagatactcatttatg attttgccaa ttttggtgtt 1740 ctcaggttat cggagccagc accgctctttgaccttgcca tgcttgcctt agatagtcca 1800 gagagtggct ggacagagga agatggtcccaaagaaggac ttgctgaata cattgttgag 1860 tttctgaaga agaaggctga gatgcttgcagactatttct ctttggaaat tgatgaggaa 1920 gggaacctga ttggattacc ccttctgattgacaactatg tgcccccttt ggagggactg 1980 cctatcttca ttcttcgact agccactgaggtgaattggg acgaagaaaa ggaatgtttt 2040 gaaagcctca gtaaagaatg cgctatgttctattccatcc ggaagcagta catatctgag 2100 gagtcgaccc tctcaggcca gcagagtgaagtgcctggct ccattccaaa ctcctggaag 2160 tggactgtgg aacacattgt ctataaagccttgcgctcac acattctgcc tcctaaacat 2220 ttcacagaag atggaaatat cctgcagcttgctaacctgc ctgatctata caaagtcttt 2280 gagaggtgtt aaatatggtt atttatgcactgtgggatgt gttcttcttt ctctgtattc 2340 cgatacaaag tgttgtatca aagtgtgatatacaaagtgt accaacataa gtgttggtag 2400 cacttaagac ttatacttgc cttctgatagtattccttta tacacagtgg attgattata 2460 aataaataga tgtgtcttaa cata 2484 12756 PRT Homo sapiens 12 Met Ser Phe Val Ala Gly Val Ile Arg Arg Leu AspGlu Thr Val Val 1 5 10 15 Asn Arg Ile Ala Ala Gly Glu Val Ile Gln ArgPro Ala Asn Ala Ile 20 25 30 Lys Glu Met Ile Glu Asn Cys Leu Asp Ala LysSer Thr Ser Ile Gln 35 40 45 Val Ile Val Lys Glu Gly Gly Leu Lys Leu IleGln Ile Gln Asp Asn 50 55 60 Gly Thr Gly Ile Arg Lys Glu Asp Leu Asp IleVal Cys Glu Arg Phe 65 70 75 80 Thr Thr Ser Lys Leu Gln Ser Phe Glu AspLeu Ala Ser Ile Ser Thr 85 90 95 Tyr Gly Phe Arg Gly Glu Ala Leu Ala SerIle Ser His Val Ala His 100 105 110 Val Thr Ile Thr Thr Lys Thr Ala AspGly Lys Cys Ala Tyr Arg Ala 115 120 125 Ser Tyr Ser Asp Gly Lys Leu LysAla Pro Pro Lys Pro Cys Ala Gly 130 135 140 Asn Gln Gly Thr Gln Ile ThrVal Glu Asp Leu Phe Tyr Asn Ile Ala 145 150 155 160 Thr Arg Arg Lys AlaLeu Lys Asn Pro Ser Glu Glu Tyr Gly Lys Ile 165 170 175 Leu Glu Val ValGly Arg Tyr Ser Val His Asn Ala Gly Ile Ser Phe 180 185 190 Ser Val LysLys Gln Gly Glu Thr Val Ala Asp Val Arg Thr Leu Pro 195 200 205 Asn AlaSer Thr Val Asp Asn Ile Arg Ser Ile Phe Gly Asn Ala Val 210 215 220 SerArg Glu Leu Ile Glu Ile Gly Cys Glu Asp Lys Thr Leu Ala Phe 225 230 235240 Lys Met Asn Gly Tyr Ile Ser Asn Ala Asn Tyr Ser Val Lys Lys Cys 245250 255 Ile Phe Leu Leu Phe Ile Asn His Arg Leu Val Glu Ser Thr Ser Leu260 265 270 Arg Lys Ala Ile Glu Thr Val Tyr Ala Ala Tyr Leu Pro Lys AsnThr 275 280 285 His Pro Phe Leu Tyr Leu Ser Leu Glu Ile Ser Pro Gln AsnVal Asp 290 295 300 Val Asn Val His Pro Thr Lys His Glu Val His Phe LeuHis Glu Glu 305 310 315 320 Ser Ile Leu Glu Arg Val Gln Gln His Ile GluSer Lys Leu Leu Gly 325 330 335 Ser Asn Ser Ser Arg Met Tyr Phe Thr GlnThr Leu Leu Pro Gly Leu 340 345 350 Ala Gly Pro Ser Gly Glu Met Val LysSer Thr Thr Ser Leu Thr Ser 355 360 365 Ser Ser Thr Ser Gly Ser Ser AspLys Val Tyr Ala His Gln Met Val 370 375 380 Arg Thr Asp Ser Arg Glu GlnLys Leu Asp Ala Phe Leu Gln Pro Leu 385 390 395 400 Ser Lys Pro Leu SerSer Gln Pro Gln Ala Ile Val Thr Glu Asp Lys 405 410 415 Thr Asp Ile SerSer Gly Arg Ala Arg Gln Gln Asp Glu Glu Met Leu 420 425 430 Glu Leu ProAla Pro Ala Glu Val Ala Ala Lys Asn Gln Ser Leu Glu 435 440 445 Gly AspThr Thr Lys Gly Thr Ser Glu Met Ser Glu Lys Arg Gly Pro 450 455 460 ThrSer Ser Asn Pro Arg Lys Arg His Arg Glu Asp Ser Asp Val Glu 465 470 475480 Met Val Glu Asp Asp Ser Arg Lys Glu Met Thr Ala Ala Cys Thr Pro 485490 495 Arg Arg Arg Ile Ile Asn Leu Thr Ser Val Leu Ser Leu Gln Glu Glu500 505 510 Ile Asn Glu Gln Gly His Glu Val Leu Arg Glu Met Leu His AsnHis 515 520 525 Ser Phe Val Gly Cys Val Asn Pro Gln Trp Ala Leu Ala GlnHis Gln 530 535 540 Thr Lys Leu Tyr Leu Leu Asn Thr Thr Lys Leu Ser GluGlu Leu Phe 545 550 555 560 Tyr Gln Ile Leu Ile Tyr Asp Phe Ala Asn PheGly Val Leu Arg Leu 565 570 575 Ser Glu Pro Ala Pro Leu Phe Asp Leu AlaMet Leu Ala Leu Asp Ser 580 585 590 Pro Glu Ser Gly Trp Thr Glu Glu AspGly Pro Lys Glu Gly Leu Ala 595 600 605 Glu Tyr Ile Val Glu Phe Leu LysLys Lys Ala Glu Met Leu Ala Asp 610 615 620 Tyr Phe Ser Leu Glu Ile AspGlu Glu Gly Asn Leu Ile Gly Leu Pro 625 630 635 640 Leu Leu Ile Asp AsnTyr Val Pro Pro Leu Glu Gly Leu Pro Ile Phe 645 650 655 Ile Leu Arg LeuAla Thr Glu Val Asn Trp Asp Glu Glu Lys Glu Cys 660 665 670 Phe Glu SerLeu Ser Lys Glu Cys Ala Met Phe Tyr Ser Ile Arg Lys 675 680 685 Gln TyrIle Ser Glu Glu Ser Thr Leu Ser Gly Gln Gln Ser Glu Val 690 695 700 ProGly Ser Ile Pro Asn Ser Trp Lys Trp Thr Val Glu His Ile Val 705 710 715720 Tyr Lys Ala Leu Arg Ser His Ile Leu Pro Pro Lys His Phe Thr Glu 725730 735 Asp Gly Asn Ile Leu Gln Leu Ala Asn Leu Pro Asp Leu Tyr Lys Val740 745 750 Phe Glu Arg Cys 755 13 426 DNA Homo sapiens 13 cgaggcggatcgggtgttgc atccatggag cgagctgaga gctcgagtac agaacctgct 60 aaggccatcaaacctattga tcggaagtca gtccatcaga tttgctctgg gcaggtggta 120 ctgagtctaagcactgcggt aaaggagtta gtagaaaaca gtctggatgc tggtgccact 180 aatattgatctaaagcttaa ggactatgga gtggatctta ttgaagtttc agacaatgga 240 tgtggggtagaagaagaaaa cttcgaaggc ttaactctga aacatcacac atctaagatt 300 caagagtttgccgacctaac tcaggttgaa acttttggct ttcgggggga agctctgagc 360 tcactttgtgcactgagcga tgtcaccatt tctacctgcc acgcatcggc gaaggttgga 420 acttga 426 14133 PRT Homo sapiens 14 Met Lys Gln Leu Pro Ala Ala Thr Val Arg Leu LeuSer Ser Ser Gln 1 5 10 15 Ile Ile Thr Ser Val Val Ser Val Val Lys GluLeu Ile Glu Asn Ser 20 25 30 Leu Asp Ala Gly Ala Thr Ser Val Asp Val LysLeu Glu Asn Tyr Gly 35 40 45 Phe Asp Lys Ile Glu Val Arg Asp Asn Gly GluGly Ile Lys Ala Val 50 55 60 Asp Ala Pro Val Met Ala Met Lys Tyr Tyr ThrSer Lys Ile Asn Ser 65 70 75 80 His Glu Asp Leu Glu Asn Leu Thr Thr TyrGly Phe Arg Gly Glu Ala 85 90 95 Leu Gly Ser Ile Cys Cys Ile Ala Glu ValLeu Ile Thr Thr Arg Thr 100 105 110 Ala Ala Asp Asn Phe Ser Thr Gln TyrVal Leu Asp Gly Ser Gly His 115 120 125 Ile Leu Ser Gln Lys 130 15 4264DNA Homo sapiens 15 atttcccgcc agcaggagcc gcgcggtaga tgcggtgcttttaggagctc cgtccgacag 60 aacggttggg ccttgccggc tgtcggtatg tcgcgacagagcaccctgta cagcttcttc 120 cccaagtctc cggcgctgag tgatgccaac aaggcctcggccagggcctc acgcgaaggc 180 ggccgtgccg ccgctgcccc cggggcctct ccttccccaggcggggatgc ggcctggagc 240 gaggctgggc ctgggcccag gcccttggcg cgatccgcgtcaccgcccaa ggcgaagaac 300 ctcaacggag ggctgcggag atcggtagcg cctgctgcccccaccagttg tgacttctca 360 ccaggagatt tggtttgggc caagatggag ggttacccctggtggccttg tctggtttac 420 aaccacccct ttgatggaac attcatccgc gagaaagggaaatcagtccg tgttcatgta 480 cagttttttg atgacagccc aacaaggggc tgggttagcaaaaggctttt aaagccatat 540 acaggttcaa aatcaaagga agcccagaag ggaggtcatttttacagtgc aaagcctgaa 600 atactgagag caatgcaacg tgcagatgaa gccttaaataaagacaagat taagaggctt 660 gaattggcag tttgtgatga gccctcagag ccagaagaggaagaagagat ggaggtaggc 720 acaacttacg taacagataa gagtgaagaa gataatgaaattgagagtga agaggaagta 780 cagcctaaga cacaaggatc taggcgaagt agccgccaaataaaaaaacg aagggtcata 840 tcagattctg agagtgacat tggtggctct gatgtggaatttaagccaga cactaaggag 900 gaaggaagca gtgatgaaat aagcagtgga gtgggggatagtgagagtga aggcctgaac 960 agccctgtca aagttgctcg aaagcggaag agaatggtgactggaaatgg ctctcttaaa 1020 aggaaaagct ctaggaagga aacgccctca gccaccaaacaagcaactag catttcatca 1080 gaaaccaaga atactttgag agctttctct gcccctcaaaattctgaatc ccaagcccac 1140 gttagtggag gtggtgatga cagtagtcgc cctactgtttggtatcatga aactttagaa 1200 tggcttaagg aggaaaagag aagagatgag cacaggaggaggcctgatca ccccgatttt 1260 gatgcatcta cactctatgt gcctgaggat ttcctcaattcttgtactcc tgggatgagg 1320 aagtggtggc agattaagtc tcagaacttt gatcttgtcatctgttacaa ggtggggaaa 1380 ttttatgagc tgtaccacat ggatgctctt attggagtcagtgaactggg gctggtattc 1440 atgaaaggca actgggccca ttctggcttt cctgaaattgcatttggccg ttattcagat 1500 tccctggtgc agaagggcta taaagtagca cgagtggaacagactgagac tccagaaatg 1560 atggaggcac gatgtagaaa gatggcacat atatccaagtatgatagagt ggtgaggagg 1620 gagatctgta ggatcattac caagggtaca cagacttacagtgtgctgga aggtgatccc 1680 tctgagaact acagtaagta tcttcttagc ctcaaagaaaaagaggaaga ttcttctggc 1740 catactcgtg catatggtgt gtgctttgtt gatacttcactgggaaagtt tttcataggt 1800 cagttttcag atgatcgcca ttgttcgaga tttaggactctagtggcaca ctatccccca 1860 gtacaagttt tatttgaaaa aggaaatctc tcaaaggaaactaaaacaat tctaaagagt 1920 tcattgtcct gttctcttca ggaaggtctg atacccggctcccagttttg ggatgcatcc 1980 aaaactttga gaactctcct tgaggaagaa tattttagggaaaagctaag tgatggcatt 2040 ggggtgatgt taccccaggt gcttaaaggt atgacttcagagtctgattc cattgggttg 2100 acaccaggag agaaaagtga attggccctc tctgctctaggtggttgtgt cttctacctc 2160 aaaaaatgcc ttattgatca ggagctttta tcaatggctaattttgaaga atatattccc 2220 ttggattctg acacagtcag cactacaaga tctggtgctatcttcaccaa agcctatcaa 2280 cgaatggtgc tagatgcagt gacattaaac aacttggagatttttctgaa tggaacaaat 2340 ggttctactg aaggaaccct actagagagg gttgatacttgccatactcc ttttggtaag 2400 cggctcctaa agcaatggct ttgtgcccca ctctgtaaccattatgctat taatgatcgt 2460 ctagatgcca tagaagacct catggttgtg cctgacaaaatctccgaagt tgtagagctt 2520 ctaaagaagc ttccagatct tgagaggcta ctcagtaaaattcataatgt tgggtctccc 2580 ctgaagagtc agaaccaccc agacagcagg gctataatgtatgaagaaac tacatacagc 2640 aagaagaaga ttattgattt tctttctgct ctggaaggattcaaagtaat gtgtaaaatt 2700 atagggatca tggaagaagt tgctgatggt tttaagtctaaaatccttaa gcaggtcatc 2760 tctctgcaga caaaaaatcc tgaaggtcgt tttcctgatttgactgtaga attgaaccga 2820 tgggatacag cctttgacca tgaaaaggct cgaaagactggacttattac tcccaaagca 2880 ggctttgact ctgattatga ccaagctctt gctgacataagagaaaatga acagagcctc 2940 ctggaatacc tagagaaaca gcgcaacaga attggctgtaggaccatagt ctattggggg 3000 attggtagga accgttacca gctggaaatt cctgagaatttcaccactcg caatttgcca 3060 gaagaatacg agttgaaatc taccaagaag ggctgtaaacgatactggac caaaactatt 3120 gaaaagaagt tggctaatct cataaatgct gaagaacggagggatgtatc attgaaggac 3180 tgcatgcggc gactgttcta taactttgat aaaaattacaaggactggca gtctgctgta 3240 gagtgtatcg cagtgttgga tgttttactg tgcctggctaactatagtcg agggggtgat 3300 ggtcctatgt gtcgcccagt aattctgttg ccggaagatacccccccctt cttagagctt 3360 aaaggatcac gccatccttg cattacgaag actttttttggagatgattt tattcctaat 3420 gacattctaa taggctgtga ggaagaggag caggaaaatggcaaagccta ttgtgtgctt 3480 gttactggac caaatatggg gggcaagtct acgcttatgagacaggctgg cttattagct 3540 gtaatggccc agatgggttg ttacgtccct gctgaagtgtgcaggctcac accaattgat 3600 agagtgttta ctagacttgg tgcctcagac agaataatgtcaggtgaaag tacatttttt 3660 gttgaattaa gtgaaactgc cagcatactc atgcatgcaacagcacattc tctggtgctt 3720 gtggatgaat taggaagagg tactgcaaca tttgatgggacggcaatagc aaatgcagtt 3780 gttaaagaac ttgctgagac tataaaatgt cgtacattattttcaactca ctaccattca 3840 ttagtagaag attattctca aaatgttgct gtgcgcctaggacatatggc atgcatggta 3900 gaaaatgaat gtgaagaccc cagccaggag actattacgttcctctataa attcattaag 3960 ggagcttgtc ctaaaagcta tggctttaat gcagcaaggcttgctaatct cccagaggaa 4020 gttattcaaa agggacatag aaaagcaaga gaatttgagaagatgaatca gtcactacga 4080 ttatttcggg aagtttgcct ggctagtgaa aggtcaactgtagatgctga agctgtccat 4140 aaattgctga ctttgattaa ggaattatag actgactacattggaagctt tgagttgact 4200 tctgaccaaa ggtggtaaat tcagacaaca ttatgatctaataaacttta ttttttaaaa 4260 atga 4264 16 1360 PRT Homo sapiens 16 Met SerArg Gln Ser Thr Leu Tyr Ser Phe Phe Pro Lys Ser Pro Ala 1 5 10 15 LeuSer Asp Ala Asn Lys Ala Ser Ala Arg Ala Ser Arg Glu Gly Gly 20 25 30 ArgAla Ala Ala Ala Pro Gly Ala Ser Pro Ser Pro Gly Gly Asp Ala 35 40 45 AlaTrp Ser Glu Ala Gly Pro Gly Pro Arg Pro Leu Ala Arg Ser Ala 50 55 60 SerPro Pro Lys Ala Lys Asn Leu Asn Gly Gly Leu Arg Arg Ser Val 65 70 75 80Ala Pro Ala Ala Pro Thr Ser Cys Asp Phe Ser Pro Gly Asp Leu Val 85 90 95Trp Ala Lys Met Glu Gly Tyr Pro Trp Trp Pro Cys Leu Val Tyr Asn 100 105110 His Pro Phe Asp Gly Thr Phe Ile Arg Glu Lys Gly Lys Ser Val Arg 115120 125 Val His Val Gln Phe Phe Asp Asp Ser Pro Thr Arg Gly Trp Val Ser130 135 140 Lys Arg Leu Leu Lys Pro Tyr Thr Gly Ser Lys Ser Lys Glu AlaGln 145 150 155 160 Lys Gly Gly His Phe Tyr Ser Ala Lys Pro Glu Ile LeuArg Ala Met 165 170 175 Gln Arg Ala Asp Glu Ala Leu Asn Lys Asp Lys IleLys Arg Leu Glu 180 185 190 Leu Ala Val Cys Asp Glu Pro Ser Glu Pro GluGlu Glu Glu Glu Met 195 200 205 Glu Val Gly Thr Thr Tyr Val Thr Asp LysSer Glu Glu Asp Asn Glu 210 215 220 Ile Glu Ser Glu Glu Glu Val Gln ProLys Thr Gln Gly Ser Arg Arg 225 230 235 240 Ser Ser Arg Gln Ile Lys LysArg Arg Val Ile Ser Asp Ser Glu Ser 245 250 255 Asp Ile Gly Gly Ser AspVal Glu Phe Lys Pro Asp Thr Lys Glu Glu 260 265 270 Gly Ser Ser Asp GluIle Ser Ser Gly Val Gly Asp Ser Glu Ser Glu 275 280 285 Gly Leu Asn SerPro Val Lys Val Ala Arg Lys Arg Lys Arg Met Val 290 295 300 Thr Gly AsnGly Ser Leu Lys Arg Lys Ser Ser Arg Lys Glu Thr Pro 305 310 315 320 SerAla Thr Lys Gln Ala Thr Ser Ile Ser Ser Glu Thr Lys Asn Thr 325 330 335Leu Arg Ala Phe Ser Ala Pro Gln Asn Ser Glu Ser Gln Ala His Val 340 345350 Ser Gly Gly Gly Asp Asp Ser Ser Arg Pro Thr Val Trp Tyr His Glu 355360 365 Thr Leu Glu Trp Leu Lys Glu Glu Lys Arg Arg Asp Glu His Arg Arg370 375 380 Arg Pro Asp His Pro Asp Phe Asp Ala Ser Thr Leu Tyr Val ProGlu 385 390 395 400 Asp Phe Leu Asn Ser Cys Thr Pro Gly Met Arg Lys TrpTrp Gln Ile 405 410 415 Lys Ser Gln Asn Phe Asp Leu Val Ile Cys Tyr LysVal Gly Lys Phe 420 425 430 Tyr Glu Leu Tyr His Met Asp Ala Leu Ile GlyVal Ser Glu Leu Gly 435 440 445 Leu Val Phe Met Lys Gly Asn Trp Ala HisSer Gly Phe Pro Glu Ile 450 455 460 Ala Phe Gly Arg Tyr Ser Asp Ser LeuVal Gln Lys Gly Tyr Lys Val 465 470 475 480 Ala Arg Val Glu Gln Thr GluThr Pro Glu Met Met Glu Ala Arg Cys 485 490 495 Arg Lys Met Ala His IleSer Lys Tyr Asp Arg Val Val Arg Arg Glu 500 505 510 Ile Cys Arg Ile IleThr Lys Gly Thr Gln Thr Tyr Ser Val Leu Glu 515 520 525 Gly Asp Pro SerGlu Asn Tyr Ser Lys Tyr Leu Leu Ser Leu Lys Glu 530 535 540 Lys Glu GluAsp Ser Ser Gly His Thr Arg Ala Tyr Gly Val Cys Phe 545 550 555 560 ValAsp Thr Ser Leu Gly Lys Phe Phe Ile Gly Gln Phe Ser Asp Asp 565 570 575Arg His Cys Ser Arg Phe Arg Thr Leu Val Ala His Tyr Pro Pro Val 580 585590 Gln Val Leu Phe Glu Lys Gly Asn Leu Ser Lys Glu Thr Lys Thr Ile 595600 605 Leu Lys Ser Ser Leu Ser Cys Ser Leu Gln Glu Gly Leu Ile Pro Gly610 615 620 Ser Gln Phe Trp Asp Ala Ser Lys Thr Leu Arg Thr Leu Leu GluGlu 625 630 635 640 Glu Tyr Phe Arg Glu Lys Leu Ser Asp Gly Ile Gly ValMet Leu Pro 645 650 655 Gln Val Leu Lys Gly Met Thr Ser Glu Ser Asp SerIle Gly Leu Thr 660 665 670 Pro Gly Glu Lys Ser Glu Leu Ala Leu Ser AlaLeu Gly Gly Cys Val 675 680 685 Phe Tyr Leu Lys Lys Cys Leu Ile Asp GlnGlu Leu Leu Ser Met Ala 690 695 700 Asn Phe Glu Glu Tyr Ile Pro Leu AspSer Asp Thr Val Ser Thr Thr 705 710 715 720 Arg Ser Gly Ala Ile Phe ThrLys Ala Tyr Gln Arg Met Val Leu Asp 725 730 735 Ala Val Thr Leu Asn AsnLeu Glu Ile Phe Leu Asn Gly Thr Asn Gly 740 745 750 Ser Thr Glu Gly ThrLeu Leu Glu Arg Val Asp Thr Cys His Thr Pro 755 760 765 Phe Gly Lys ArgLeu Leu Lys Gln Trp Leu Cys Ala Pro Leu Cys Asn 770 775 780 His Tyr AlaIle Asn Asp Arg Leu Asp Ala Ile Glu Asp Leu Met Val 785 790 795 800 ValPro Asp Lys Ile Ser Glu Val Val Glu Leu Leu Lys Lys Leu Pro 805 810 815Asp Leu Glu Arg Leu Leu Ser Lys Ile His Asn Val Gly Ser Pro Leu 820 825830 Lys Ser Gln Asn His Pro Asp Ser Arg Ala Ile Met Tyr Glu Glu Thr 835840 845 Thr Tyr Ser Lys Lys Lys Ile Ile Asp Phe Leu Ser Ala Leu Glu Gly850 855 860 Phe Lys Val Met Cys Lys Ile Ile Gly Ile Met Glu Glu Val AlaAsp 865 870 875 880 Gly Phe Lys Ser Lys Ile Leu Lys Gln Val Ile Ser LeuGln Thr Lys 885 890 895 Asn Pro Glu Gly Arg Phe Pro Asp Leu Thr Val GluLeu Asn Arg Trp 900 905 910 Asp Thr Ala Phe Asp His Glu Lys Ala Arg LysThr Gly Leu Ile Thr 915 920 925 Pro Lys Ala Gly Phe Asp Ser Asp Tyr AspGln Ala Leu Ala Asp Ile 930 935 940 Arg Glu Asn Glu Gln Ser Leu Leu GluTyr Leu Glu Lys Gln Arg Asn 945 950 955 960 Arg Ile Gly Cys Arg Thr IleVal Tyr Trp Gly Ile Gly Arg Asn Arg 965 970 975 Tyr Gln Leu Glu Ile ProGlu Asn Phe Thr Thr Arg Asn Leu Pro Glu 980 985 990 Glu Tyr Glu Leu LysSer Thr Lys Lys Gly Cys Lys Arg Tyr Trp Thr 995 1000 1005 Lys Thr IleGlu Lys Lys Leu Ala Asn Leu Ile Asn Ala Glu Glu 1010 1015 1020 Arg ArgAsp Val Ser Leu Lys Asp Cys Met Arg Arg Leu Phe Tyr 1025 1030 1035 AsnPhe Asp Lys Asn Tyr Lys Asp Trp Gln Ser Ala Val Glu Cys 1040 1045 1050Ile Ala Val Leu Asp Val Leu Leu Cys Leu Ala Asn Tyr Ser Arg 1055 10601065 Gly Gly Asp Gly Pro Met Cys Arg Pro Val Ile Leu Leu Pro Glu 10701075 1080 Asp Thr Pro Pro Phe Leu Glu Leu Lys Gly Ser Arg His Pro Cys1085 1090 1095 Ile Thr Lys Thr Phe Phe Gly Asp Asp Phe Ile Pro Asn AspIle 1100 1105 1110 Leu Ile Gly Cys Glu Glu Glu Glu Gln Glu Asn Gly LysAla Tyr 1115 1120 1125 Cys Val Leu Val Thr Gly Pro Asn Met Gly Gly LysSer Thr Leu 1130 1135 1140 Met Arg Gln Ala Gly Leu Leu Ala Val Met AlaGln Met Gly Cys 1145 1150 1155 Tyr Val Pro Ala Glu Val Cys Arg Leu ThrPro Ile Asp Arg Val 1160 1165 1170 Phe Thr Arg Leu Gly Ala Ser Asp ArgIle Met Ser Gly Glu Ser 1175 1180 1185 Thr Phe Phe Val Glu Leu Ser GluThr Ala Ser Ile Leu Met His 1190 1195 1200 Ala Thr Ala His Ser Leu ValLeu Val Asp Glu Leu Gly Arg Gly 1205 1210 1215 Thr Ala Thr Phe Asp GlyThr Ala Ile Ala Asn Ala Val Val Lys 1220 1225 1230 Glu Leu Ala Glu ThrIle Lys Cys Arg Thr Leu Phe Ser Thr His 1235 1240 1245 Tyr His Ser LeuVal Glu Asp Tyr Ser Gln Asn Val Ala Val Arg 1250 1255 1260 Leu Gly HisMet Ala Cys Met Val Glu Asn Glu Cys Glu Asp Pro 1265 1270 1275 Ser GlnGlu Thr Ile Thr Phe Leu Tyr Lys Phe Ile Lys Gly Ala 1280 1285 1290 CysPro Lys Ser Tyr Gly Phe Asn Ala Ala Arg Leu Ala Asn Leu 1295 1300 1305Pro Glu Glu Val Ile Gln Lys Gly His Arg Lys Ala Arg Glu Phe 1310 13151320 Glu Lys Met Asn Gln Ser Leu Arg Leu Phe Arg Glu Val Cys Leu 13251330 1335 Ala Ser Glu Arg Ser Thr Val Asp Ala Glu Ala Val His Lys Leu1340 1345 1350 Leu Thr Leu Ile Lys Glu Leu 1355 1360 17 1408 DNA Homosapiens 17 ggcgctccta cctgcaagtg gctagtgcca agtgctgggc cgccgctcctgccgtgcatg 60 ttggggagcc agtacatgca ggtgggctcc acacggagag gggcgcagacccggtgacag 120 ggctttacct ggtacatcgg catggcgcaa ccaaagcaag agagggtggcgcgtgccaga 180 caccaacggt cggaaaccgc cagacaccaa cggtcggaaa ccgccaagacaccaacgctc 240 ggaaaccgcc agacaccaac gctcggaaac cgccagacac caaggctcggaatccacgcc 300 aggccacgac ggagggcgac tacctccctt ctgaccctgc tgctggcgttcggaaaaaac 360 gcagtccggt gtgctctgat tggtccaggc tctttgacgt cacggactcgacctttgaca 420 gagccactag gcgaaaagga gagacgggaa gtattttttc cgccccgcccggaaagggtg 480 gagcacaacg tcgaaagcag ccgttgggag cccaggaggc ggggcgcctgtgggagccgt 540 ggagggaact ttcccagtcc ccgaggcgga tccggtgttg catccttggagcgagctgag 600 aactcgagta cagaacctgc taaggccatc aaacctattg atcggaagtcagtccatcag 660 atttgctctg ggccggtggt accgagtcta aggccgaatg cggtgaaggagttagtagaa 720 aacagtctgg atgctggtgc cactaatgtt gatctaaagc ttaaggactatggagtggat 780 ctcattgaag tttcaggcaa tggatgtggg gtagaagaag aaaacttcgaaggctttact 840 ctgaaacatc acacatgtaa gattcaagag tttgccgacc taactcaggtggaaactttt 900 ggctttcggg gggaagctct gagctcactt tgtgcactga gtgatgtcaccatttctacc 960 tgccgtgtat cagcgaaggt tgggactcga ctggtgtttg atcactatgggaaaatcatc 1020 cagaaaaccc cctacccccg ccccagaggg atgacagtca gcgtgaagcagttattttct 1080 acgctacctg tgcaccataa agaatttcaa aggaatatta agaagaaacgtgcctgcttc 1140 cccttcgcct tctgccgtga ttgtcagttt cctgaggcct ccccagccatgcttcctgta 1200 cagcctgtag aactgactcc tagaagtacc ccaccccacc cctgctccttggaggacaac 1260 gtgatcactg tattcagctc tgtcaagaat ggtccaggtt cttctagatgatctgcacaa 1320 atggttcctc tcctccttcc tgatgtctgc cattagcatt ggaataaagttcctgctgaa 1380 aatccaaaaa aaaaaaaaaa aaaaaaaa 1408 18 389 PRT Homosapiens 18 Met Ala Gln Pro Lys Gln Glu Arg Val Ala Arg Ala Arg His GlnArg 1 5 10 15 Ser Glu Thr Ala Arg His Gln Arg Ser Glu Thr Ala Lys ThrPro Thr 20 25 30 Leu Gly Asn Arg Gln Thr Pro Thr Leu Gly Asn Arg Gln ThrPro Arg 35 40 45 Leu Gly Ile His Ala Arg Pro Arg Arg Arg Ala Thr Thr SerLeu Leu 50 55 60 Thr Leu Leu Leu Ala Phe Gly Lys Asn Ala Val Arg Cys AlaLeu Ile 65 70 75 80 Gly Pro Gly Ser Leu Thr Ser Arg Thr Arg Pro Leu ThrGlu Pro Leu 85 90 95 Gly Glu Lys Glu Arg Arg Glu Val Phe Phe Pro Pro ArgPro Glu Arg 100 105 110 Val Glu His Asn Val Glu Ser Ser Arg Trp Glu ProArg Arg Arg Gly 115 120 125 Ala Cys Gly Ser Arg Gly Gly Asn Phe Pro SerPro Arg Gly Gly Ser 130 135 140 Gly Val Ala Ser Leu Glu Arg Ala Glu AsnSer Ser Thr Glu Pro Ala 145 150 155 160 Lys Ala Ile Lys Pro Ile Asp ArgLys Ser Val His Gln Ile Cys Ser 165 170 175 Gly Pro Val Val Pro Ser LeuArg Pro Asn Ala Val Lys Glu Leu Val 180 185 190 Glu Asn Ser Leu Asp AlaGly Ala Thr Asn Val Asp Leu Lys Leu Lys 195 200 205 Asp Tyr Gly Val AspLeu Ile Glu Val Ser Gly Asn Gly Cys Gly Val 210 215 220 Glu Glu Glu AsnPhe Glu Gly Phe Thr Leu Lys His His Thr Cys Lys 225 230 235 240 Ile GlnGlu Phe Ala Asp Leu Thr Gln Val Glu Thr Phe Gly Phe Arg 245 250 255 GlyGlu Ala Leu Ser Ser Leu Cys Ala Leu Ser Asp Val Thr Ile Ser 260 265 270Thr Cys Arg Val Ser Ala Lys Val Gly Thr Arg Leu Val Phe Asp His 275 280285 Tyr Gly Lys Ile Ile Gln Lys Thr Pro Tyr Pro Arg Pro Arg Gly Met 290295 300 Thr Val Ser Val Lys Gln Leu Phe Ser Thr Leu Pro Val His His Lys305 310 315 320 Glu Phe Gln Arg Asn Ile Lys Lys Lys Arg Ala Cys Phe ProPhe Ala 325 330 335 Phe Cys Arg Asp Cys Gln Phe Pro Glu Ala Ser Pro AlaMet Leu Pro 340 345 350 Val Gln Pro Val Glu Leu Thr Pro Arg Ser Thr ProPro His Pro Cys 355 360 365 Ser Leu Glu Asp Asn Val Ile Thr Val Phe SerSer Val Lys Asn Gly 370 375 380 Pro Gly Ser Ser Arg 385 19 1785 DNA Homosapiens 19 tttttagaaa ctgatgttta ttttccatca accatttttc catgctgcttaagagaatat 60 gcaagaacag cttaagacca gtcagtggtt gctcctaccc attcagtggcctgagcagtg 120 gggagctgca gaccagtctt ccgtggcagg ctgagcgctc cagtcttcagtagggaattg 180 ctgaataggc acagagggca cctgtacacc ttcagaccag tctgcaacctcaggctgagt 240 agcagtgaac tcaggagcgg gagcagtcca ttcaccctga aattcctccttggtcactgc 300 cttctcagca gcagcctgct cttctttttc aatctcttca ggatctctgtagaagtacag 360 atcaggcatg acctcccatg ggtgttcacg ggaaatggtg ccacgcatgcgcagaacttc 420 ccgagccagc atccaccaca ttaaacccac tgagtgagct cccttgttgttgcatgggat 480 ggcaatgtcc acatagcgca gaggagaatc tgtgttacac agcgcaatggtaggtaggtt 540 aacataagat gcctccgtga gaggcgaagg ggcggcggga cccgggcctggcccgtatgt 600 gtccttggcg gcctagacta ggccgtcgct gtatggtgag ccccagggaggcggatctgg 660 gcccccagaa ggacacccgc ctggatttgc cccgtagccc ggcccgggcccctcgggagc 720 agaacagcct tggtgaggtg gacaggaggg gacctcgcga gcagacgcgcgcgccagcga 780 cagcagcccc gccccggcct ctcgggagcc ggggggcaga ggctgcggagccccaggagg 840 gtctatcagc cacagtctct gcatgtttcc aagagcaaca ggaaatgaacacattgcagg 900 ggccagtgtc attcaaagat gtggctgtgg atttcaccca ggaggagtggcggcaactgg 960 accctgatga gaagatagca tacggggatg tgatgttgga gaactacagccatctagttt 1020 ctgtggggta tgattatcac caagccaaac atcatcatgg agtggaggtgaaggaagtgg 1080 agcagggaga ggagccgtgg ataatggaag gtgaatttcc atgtcaacatagtccagaac 1140 ctgctaaggc catcaaacct attgatcgga agtcagtcca tcagatttgctctgggccag 1200 tggtactgag tctaagcact gcagtgaagg agttagtaga aaacagtctggatgctggtg 1260 ccactaatat tgatctaaag cttaaggact atggagtgga tctcattgaagtttcagaca 1320 atggatgtgg ggtagaagaa gaaaactttg aaggcttaat ctctttcagctctgaaacat 1380 cacacatgta agattcaaga gtttgccgac ctaactgaag ttgaaactttcggttttcag 1440 ggggaagctc tgagctcact gtgtgcactg agcgatgtca ccatttctacctgccacgcg 1500 ttggtgaagg ttgggactcg actggtgttt gatcacgatg ggaaaatcatccaggaaacc 1560 ccctaccccc accccagagg gaccacagtc agcgtgaagc agttattttctacgctacct 1620 gtgcgccata aggaatttca aaggaatatt aagaagacgt gcctgcttccccttcgcctt 1680 ctgccgtgat tgtcagtttc ctgaggcctc cccagccatg cttcctgtacagcctgcaga 1740 actgtgagtc aattaaacct cttttcttca taaattaaaa aaaaa 178520 264 PRT Homo sapiens 20 Met Cys Pro Trp Arg Pro Arg Leu Gly Arg ArgCys Met Val Ser Pro 1 5 10 15 Arg Glu Ala Asp Leu Gly Pro Gln Lys AspThr Arg Leu Asp Leu Pro 20 25 30 Arg Ser Pro Ala Arg Ala Pro Arg Glu GlnAsn Ser Leu Gly Glu Val 35 40 45 Asp Arg Arg Gly Pro Arg Glu Gln Thr ArgAla Pro Ala Thr Ala Ala 50 55 60 Pro Pro Arg Pro Leu Gly Ser Arg Gly AlaGlu Ala Ala Glu Pro Gln 65 70 75 80 Glu Gly Leu Ser Ala Thr Val Ser AlaCys Phe Gln Glu Gln Gln Glu 85 90 95 Met Asn Thr Leu Gln Gly Pro Val SerPhe Lys Asp Val Ala Val Asp 100 105 110 Phe Thr Gln Glu Glu Trp Arg GlnLeu Asp Pro Asp Glu Lys Ile Ala 115 120 125 Tyr Gly Asp Val Met Leu GluAsn Tyr Ser His Leu Val Ser Val Gly 130 135 140 Tyr Asp Tyr His Gln AlaLys His His His Gly Val Glu Val Lys Glu 145 150 155 160 Val Glu Gln GlyGlu Glu Pro Trp Ile Met Glu Gly Glu Phe Pro Cys 165 170 175 Gln His SerPro Glu Pro Ala Lys Ala Ile Lys Pro Ile Asp Arg Lys 180 185 190 Ser ValHis Gln Ile Cys Ser Gly Pro Val Val Leu Ser Leu Ser Thr 195 200 205 AlaVal Lys Glu Leu Val Glu Asn Ser Leu Asp Ala Gly Ala Thr Asn 210 215 220Ile Asp Leu Lys Leu Lys Asp Tyr Gly Val Asp Leu Ile Glu Val Ser 225 230235 240 Asp Asn Gly Cys Gly Val Glu Glu Glu Asn Phe Glu Gly Leu Ile Ser245 250 255 Phe Ser Ser Glu Thr Ser His Met 260 21 795 DNA Homo sapiens21 atgtgtcctt ggcggcctag actaggccgt cgctgtatgg tgagccccag ggaggcggat 60ctgggccccc agaaggacac ccgcctggat ttgccccgta gcccggcccg ggcccctcgg 120gagcagaaca gccttggtga ggtggacagg aggggacctc gcgagcagac gcgcgcgcca 180gcgacagcag ccccgccccg gcctctcggg agccgggggg cagaggctgc ggagccccag 240gagggtctat cagccacagt ctctgcatgt ttccaagagc aacaggaaat gaacacattg 300caggggccag tgtcattcaa agatgtggct gtggatttca cccaggagga gtggcggcaa 360ctggaccctg atgagaagat agcatacggg gatgtgatgt tggagaacta cagccatcta 420gtttctgtgg ggtatgatta tcaccaagcc aaacatcatc atggagtgga ggtgaaggaa 480gtggagcagg gagaggagcc gtggataatg gaaggtgaat ttccatgtca acatagtcca 540gaacctgcta aggccatcaa acctattgat cggaagtcag tccatcagat ttgctctggg 600ccagtggtac tgagtctaag cactgcagtg aaggagttag tagaaaacag tctggatgct 660ggtgccacta atattgatct aaagcttaag gactatggag tggatctcat tgaagtttca 720gacaatggat gtggggtaga agaagaaaac tttgaaggct taatctcttt cagctctgaa 780acatcacaca tgtaa 795 22 264 PRT Homo sapiens 22 Met Cys Pro Trp Arg ProArg Leu Gly Arg Arg Cys Met Val Ser Pro 1 5 10 15 Arg Glu Ala Asp LeuGly Pro Gln Lys Asp Thr Arg Leu Asp Leu Pro 20 25 30 Arg Ser Pro Ala ArgAla Pro Arg Glu Gln Asn Ser Leu Gly Glu Val 35 40 45 Asp Arg Arg Gly ProArg Glu Gln Thr Arg Ala Pro Ala Thr Ala Ala 50 55 60 Pro Pro Arg Pro LeuGly Ser Arg Gly Ala Glu Ala Ala Glu Pro Gln 65 70 75 80 Glu Gly Leu SerAla Thr Val Ser Ala Cys Phe Gln Glu Gln Gln Glu 85 90 95 Met Asn Thr LeuGln Gly Pro Val Ser Phe Lys Asp Val Ala Val Asp 100 105 110 Phe Thr GlnGlu Glu Trp Arg Gln Leu Asp Pro Asp Glu Lys Ile Ala 115 120 125 Tyr GlyAsp Val Met Leu Glu Asn Tyr Ser His Leu Val Ser Val Gly 130 135 140 TyrAsp Tyr His Gln Ala Lys His His His Gly Val Glu Val Lys Glu 145 150 155160 Val Glu Gln Gly Glu Glu Pro Trp Ile Met Glu Gly Glu Phe Pro Cys 165170 175 Gln His Ser Pro Glu Pro Ala Lys Ala Ile Lys Pro Ile Asp Arg Lys180 185 190 Ser Val His Gln Ile Cys Ser Gly Pro Val Val Leu Ser Leu SerThr 195 200 205 Ala Val Lys Glu Leu Val Glu Asn Ser Leu Asp Ala Gly AlaThr Asn 210 215 220 Ile Asp Leu Lys Leu Lys Asp Tyr Gly Val Asp Leu IleGlu Val Ser 225 230 235 240 Asp Asn Gly Cys Gly Val Glu Glu Glu Asn PheGlu Gly Leu Ile Ser 245 250 255 Phe Ser Ser Glu Thr Ser His Met 260 2330 DNA Artificial Sequence synthetic oligonucleotide primer 23acgcatatgg agcgagctga gagctcgagt 30 24 75 DNA Artificial Sequencesynthetic oligonucleotide primer 24 gaattcttat cacgtagaat cgagaccgaggagagggtta gggataggct taccagttcc 60 aaccttcgcc gatgc 75 25 27 DNAArtificial Sequence synthetic oligonucleotide primer 25 acgcatatgtgtccttggcg gcctaga 27 26 75 DNA Artificial Sequence syntheticoligonucleotide primer 26 gaattcttat tacgtagaat cgagaccgag gagagggttagggataggct tacccatgtg 60 tgatgtttca gagct 75 27 49 DNA ArtificialSequence synthetic oligonucleotide primer 27 tttaatacga ctcactatagggagaccacc atggnnnnnn nnnnnnnnn 49 28 4290 DNA Homo sapiens 28atgatcaagt gcttgtcagt tgaagtacaa gccaaattgc gttctggttt ggccataagc 60tccttgggcc aatgtgttga ggaacttgcc ctcaacagta ttgatgctga agcaaaatgt 120gtggctgtca gggtgaatat ggaaaccttc caagttcaag tgatagacaa tggatttggg 180atggggagtg atgatgtaga gaaagtggga aatcgttatt tcaccagtaa atgccactcg 240gtacaggact tggagaatcc aaggttttat ggtttccgag gagaggcctt ggcaaatatt 300gctgacatgg ccagtgctgt ggaaatttcg tccaagaaaa acaggacaat gaaaactttt 360gtgaaactgt ttcagagtgg aaaagccctg aaagcttgtg aagctgatgt gactagagca 420agcgctggga ctactgtaac agtgtataac ctattttacc agcttcctgt aaggaggaaa 480tgcatggacc ctagactgga gtttgagaag gttaggcaga gaatagaagc tctctcactc 540atgcaccctt ccatttcttt ctctttgaga aatgatgttt ctggttccat ggttcttcag 600ctccctaaaa ccaaagacgt atgttcccga ttttgtcaaa tttatggatt gggaaagtcc 660caaaagctaa gagaaataag ttttaaatat aaagagtttg agcttagtgg ctatatcagc 720tctgaagcac attacaacaa gaatatgcag tttttgtttg tgaacaaaag actagtttta 780aggacaaagc tacataaact cattgacttt ttattaagga aagaaagtat tatatgcaag 840ccaaagaatg gtcccaccag taggcaaatg aattcaagtc ttcggcaccg gtctacccca 900gaactctatg gcatatatgt aattaatgtg cagtgccaat tctgtgagta tgatgtgtgc 960atggagccag ccaaaactct gattgaattt cagaactggg acactctctt gttttgcatt 1020caggaaggag tgaaaatgtt tttaaagcaa gaaaaattat ttgtggaatt atcaggtgag 1080gatattaagg aatttagtga agataatggt tttagtttat ttgatgctac tcttcagaag 1140cgtgtgactt ccgatgagag gagcaatttc caggaagcat gtaataatat tttagattcc 1200tatgagatgt ttaatttgca gtcaaaagct gtgaaaagaa aaactactgc agaaaacgta 1260aacacacaga gttctaggga ttcagaagct accagaaaaa atacaaatga tgcatttttg 1320tacatttatg aatcaggtgg tccaggccat agcaaaatga cagagccatc tttacaaaac 1380aaagacagct cttgctcaga atcaaagatg ttagaacaag agacaattgt agcatcagaa 1440gctggagaaa atgagaaaca taaaaaatct ttcctggaac atagctcttt agaaaatccg 1500tgtggaacca gtttagaaat gtttttaagc ccttttcaga caccatgtca ctttgaggag 1560agtgggcagg atctagaaat atggaaagaa agtactactg ttaatggcat ggctgccaac 1620atcttgaaaa ataatagaat tcagaatcaa ccaaagagat ttaaagatgc tactgaagtg 1680ggatgccagc ctctgccttt tgcaacaaca ttatggggag tacatagtgc tcagacagag 1740aaagagaaaa aaaaagaatc tagcaattgt ggaagaagaa atgtttttag ttatgggcga 1800gttaaattat gttccactgg ctttataact catgtagtac aaaatgaaaa aactaaatca 1860actgaaacag aacattcatt taaaaattat gttagacctg gtcccacacg tgcccaagaa 1920acatttggaa atagaacacg tcattcagtt gaaactccag acatcaaaga tttagccagc 1980actttaagta aagaatctgg tcaattgccc aacaaaaaaa attgcagaac gaatataagt 2040tatgggctag agaatgaacc tacagcaact tatacaatgt tttctgcttt tcaggaaggt 2100agcaaaaaat cacaaacaga ttgcatatta tctgatacat ccccctcttt cccctggtat 2160agacacgttt ccaatgatag taggaaaaca gataaattaa ttggtttctc caaaccaatc 2220gtccgtaaga agctaagctt gagttcacag ctaggatctt tagagaagtt taagaggcaa 2280tatgggaagg ttgaaaatcc tctggataca gaagtagagg aaagtaatgg agtcactacc 2340aatctcagtc ttcaagttga acctgacatt ctgctgaagg acaagaaccg cttagagaac 2400tctgatgttt gtaaaatcac tactatggag catagtgatt cagatagtag ttgtcaacca 2460gcaagccaca tccttgactc agagaagttt ccattctcca aggatgaaga ttgtttagaa 2520caacagatgc ttagtttgag agaaagtcct atgaccctga aggagttatc tctctttaat 2580agaaaacctt tggaccttga gaagtcatct gaatcactag cctctaaatt atccagactg 2640aagggttccg aaagagaaac tcaaacaatg gggatgatga gtcgttttaa tgaacttcca 2700aattcagatt ccagtaggaa agacagcaag ttgtgcagtg tgttaacaca agatttttgt 2760atgttattta acaacaagca tgaaaaaaca gagaatggtg tcatcccaac atcagattct 2820gccacacagg ataattcctt taataaaaat agtaaaacac attctaacag caatacaaca 2880gagaactgtg tgatatcaga aactcctttg gtattgccct ataataattc taaagttacc 2940ggtaaagatt cagatgttct tatcagagcc tcagaacaac agataggaag tcttgactct 3000cccagtggaa tgttaatgaa tccggtagaa gatgccacag gtgaccaaaa tggaatttgt 3060tttcagagtg aggaatctaa agcaagagct tgttctgaaa ctgaagagtc aaacacgtgt 3120tgttcagatt ggcagcggca tttcgatgta gccctgggaa gaatggttta tgtcaacaaa 3180atgactggac tcagcacatt cattgcccca actgaggaca ttcaggctgc ttgtactaaa 3240gacctgacaa ctgtggctgt ggatgttgta cttgagaatg ggtctcagta caggtgtcaa 3300ccttttagaa gcgaccttgt tcttcctttc cttccgagag ctcgagcaga gaggactgtg 3360atgagacagg ataacagaga tactgtggat gatactgtta gtagcgaatc gcttcagtct 3420ttgttctcag aatgggacaa tccagtattt gcccgttatc cagaggttgc tgttgatgta 3480agcagtggcc aggctgagag cttagcagtt aaaattcaca acatcttgta tccctatcgt 3540ttcaccaaag gaatgattca ttcaatgcag gttctccagc aagtagataa caagtttatt 3600gcctgtttga tgagcactaa gactgaagag aatggcgagg cagattccta cgagaagcaa 3660caggcacaag gctctggtcg gaaaaaatta ctgtcttcta ctctaattcc tccgctagag 3720ataacagtga cagaggaaca aaggagactc ttatggtgtt accacaaaaa tctggaagat 3780ctgggccttg aatttgtatt tccagacact agtgattctc tggtccttgt gggaaaagta 3840ccactatgtt ttgtggaaag agaagccaat gaacttcgga gaggaagatc tactgtgacc 3900aagagtattg tggaggaatt tatccgagaa caactggagc tactccagac caccggaggc 3960atccaaggga cattgccact gactgtccag aaggtgttgg catcccaagc ctgccatggg 4020gccattaagt ttaatgatgg cctgagctta caggaaagtt gccgccttat tgaagctctg 4080tcctcatgcc agctgccatt ccagtgtgct cacgggagac cttctatgct gccgttagct 4140gacatagacc acttggaaca ggaaaaacag attaaaccca acctcactaa acttcgcaaa 4200atggcccagg cctggcgtct ctttggaaaa gcagagtgtg atacaaggca gagcctgcag 4260caatccatgc ctccctgtga gccaccatga 4290 29 1429 PRT Homo sapiens 29 MetIle Lys Cys Leu Ser Val Glu Val Gln Ala Lys Leu Arg Ser Gly 1 5 10 15Leu Ala Ile Ser Ser Leu Gly Gln Cys Val Glu Glu Leu Ala Leu Asn 20 25 30Ser Ile Asp Ala Glu Ala Lys Cys Val Ala Val Arg Val Asn Met Glu 35 40 45Thr Phe Gln Val Gln Val Ile Asp Asn Gly Phe Gly Met Gly Ser Asp 50 55 60Asp Val Glu Lys Val Gly Asn Arg Tyr Phe Thr Ser Lys Cys His Ser 65 70 7580 Val Gln Asp Leu Glu Asn Pro Arg Phe Tyr Gly Phe Arg Gly Glu Ala 85 9095 Leu Ala Asn Ile Ala Asp Met Ala Ser Ala Val Glu Ile Ser Ser Lys 100105 110 Lys Asn Arg Thr Met Lys Thr Phe Val Lys Leu Phe Gln Ser Gly Lys115 120 125 Ala Leu Lys Ala Cys Glu Ala Asp Val Thr Arg Ala Ser Ala GlyThr 130 135 140 Thr Val Thr Val Tyr Asn Leu Phe Tyr Gln Leu Pro Val ArgArg Lys 145 150 155 160 Cys Met Asp Pro Arg Leu Glu Phe Glu Lys Val ArgGln Arg Ile Glu 165 170 175 Ala Leu Ser Leu Met His Pro Ser Ile Ser PheSer Leu Arg Asn Asp 180 185 190 Val Ser Gly Ser Met Val Leu Gln Leu ProLys Thr Lys Asp Val Cys 195 200 205 Ser Arg Phe Cys Gln Ile Tyr Gly LeuGly Lys Ser Gln Lys Leu Arg 210 215 220 Glu Ile Ser Phe Lys Tyr Lys GluPhe Glu Leu Ser Gly Tyr Ile Ser 225 230 235 240 Ser Glu Ala His Tyr AsnLys Asn Met Gln Phe Leu Phe Val Asn Lys 245 250 255 Arg Leu Val Leu ArgThr Lys Leu His Lys Leu Ile Asp Phe Leu Leu 260 265 270 Arg Lys Glu SerIle Ile Cys Lys Pro Lys Asn Gly Pro Thr Ser Arg 275 280 285 Gln Met AsnSer Ser Leu Arg His Arg Ser Thr Pro Glu Leu Tyr Gly 290 295 300 Ile TyrVal Ile Asn Val Gln Cys Gln Phe Cys Glu Tyr Asp Val Cys 305 310 315 320Met Glu Pro Ala Lys Thr Leu Ile Glu Phe Gln Asn Trp Asp Thr Leu 325 330335 Leu Phe Cys Ile Gln Glu Gly Val Lys Met Phe Leu Lys Gln Glu Lys 340345 350 Leu Phe Val Glu Leu Ser Gly Glu Asp Ile Lys Glu Phe Ser Glu Asp355 360 365 Asn Gly Phe Ser Leu Phe Asp Ala Thr Leu Gln Lys Arg Val ThrSer 370 375 380 Asp Glu Arg Ser Asn Phe Gln Glu Ala Cys Asn Asn Ile LeuAsp Ser 385 390 395 400 Tyr Glu Met Phe Asn Leu Gln Ser Lys Ala Val LysArg Lys Thr Thr 405 410 415 Ala Glu Asn Val Asn Thr Gln Ser Ser Arg AspSer Glu Ala Thr Arg 420 425 430 Lys Asn Thr Asn Asp Ala Phe Leu Tyr IleTyr Glu Ser Gly Gly Pro 435 440 445 Gly His Ser Lys Met Thr Glu Pro SerLeu Gln Asn Lys Asp Ser Ser 450 455 460 Cys Ser Glu Ser Lys Met Leu GluGln Glu Thr Ile Val Ala Ser Glu 465 470 475 480 Ala Gly Glu Asn Glu LysHis Lys Lys Ser Phe Leu Glu His Ser Ser 485 490 495 Leu Glu Asn Pro CysGly Thr Ser Leu Glu Met Phe Leu Ser Pro Phe 500 505 510 Gln Thr Pro CysHis Phe Glu Glu Ser Gly Gln Asp Leu Glu Ile Trp 515 520 525 Lys Glu SerThr Thr Val Asn Gly Met Ala Ala Asn Ile Leu Lys Asn 530 535 540 Asn ArgIle Gln Asn Gln Pro Lys Arg Phe Lys Asp Ala Thr Glu Val 545 550 555 560Gly Cys Gln Pro Leu Pro Phe Ala Thr Thr Leu Trp Gly Val His Ser 565 570575 Ala Gln Thr Glu Lys Glu Lys Lys Lys Glu Ser Ser Asn Cys Gly Arg 580585 590 Arg Asn Val Phe Ser Tyr Gly Arg Val Lys Leu Cys Ser Thr Gly Phe595 600 605 Ile Thr His Val Val Gln Asn Glu Lys Thr Lys Ser Thr Glu ThrGlu 610 615 620 His Ser Phe Lys Asn Tyr Val Arg Pro Gly Pro Thr Arg AlaGln Glu 625 630 635 640 Thr Phe Gly Asn Arg Thr Arg His Ser Val Glu ThrPro Asp Ile Lys 645 650 655 Asp Leu Ala Ser Thr Leu Ser Lys Glu Ser GlyGln Leu Pro Asn Lys 660 665 670 Lys Asn Cys Arg Thr Asn Ile Ser Tyr GlyLeu Glu Asn Glu Pro Thr 675 680 685 Ala Thr Tyr Thr Met Phe Ser Ala PheGln Glu Gly Ser Lys Lys Ser 690 695 700 Gln Thr Asp Cys Ile Leu Ser AspThr Ser Pro Ser Phe Pro Trp Tyr 705 710 715 720 Arg His Val Ser Asn AspSer Arg Lys Thr Asp Lys Leu Ile Gly Phe 725 730 735 Ser Lys Pro Ile ValArg Lys Lys Leu Ser Leu Ser Ser Gln Leu Gly 740 745 750 Ser Leu Glu LysPhe Lys Arg Gln Tyr Gly Lys Val Glu Asn Pro Leu 755 760 765 Asp Thr GluVal Glu Glu Ser Asn Gly Val Thr Thr Asn Leu Ser Leu 770 775 780 Gln ValGlu Pro Asp Ile Leu Leu Lys Asp Lys Asn Arg Leu Glu Asn 785 790 795 800Ser Asp Val Cys Lys Ile Thr Thr Met Glu His Ser Asp Ser Asp Ser 805 810815 Ser Cys Gln Pro Ala Ser His Ile Leu Asp Ser Glu Lys Phe Pro Phe 820825 830 Ser Lys Asp Glu Asp Cys Leu Glu Gln Gln Met Leu Ser Leu Arg Glu835 840 845 Ser Pro Met Thr Leu Lys Glu Leu Ser Leu Phe Asn Arg Lys ProLeu 850 855 860 Asp Leu Glu Lys Ser Ser Glu Ser Leu Ala Ser Lys Leu SerArg Leu 865 870 875 880 Lys Gly Ser Glu Arg Glu Thr Gln Thr Met Gly MetMet Ser Arg Phe 885 890 895 Asn Glu Leu Pro Asn Ser Asp Ser Ser Arg LysAsp Ser Lys Leu Cys 900 905 910 Ser Val Leu Thr Gln Asp Phe Cys Met LeuPhe Asn Asn Lys His Glu 915 920 925 Lys Thr Glu Asn Gly Val Ile Pro ThrSer Asp Ser Ala Thr Gln Asp 930 935 940 Asn Ser Phe Asn Lys Asn Ser LysThr His Ser Asn Ser Asn Thr Thr 945 950 955 960 Glu Asn Cys Val Ile SerGlu Thr Pro Leu Val Leu Pro Tyr Asn Asn 965 970 975 Ser Lys Val Thr GlyLys Asp Ser Asp Val Leu Ile Arg Ala Ser Glu 980 985 990 Gln Gln Ile GlySer Leu Asp Ser Pro Ser Gly Met Leu Met Asn Pro 995 1000 1005 Val GluAsp Ala Thr Gly Asp Gln Asn Gly Ile Cys Phe Gln Ser 1010 1015 1020 GluGlu Ser Lys Ala Arg Ala Cys Ser Glu Thr Glu Glu Ser Asn 1025 1030 1035Thr Cys Cys Ser Asp Trp Gln Arg His Phe Asp Val Ala Leu Gly 1040 10451050 Arg Met Val Tyr Val Asn Lys Met Thr Gly Leu Ser Thr Phe Ile 10551060 1065 Ala Pro Thr Glu Asp Ile Gln Ala Ala Cys Thr Lys Asp Leu Thr1070 1075 1080 Thr Val Ala Val Asp Val Val Leu Glu Asn Gly Ser Gln TyrArg 1085 1090 1095 Cys Gln Pro Phe Arg Ser Asp Leu Val Leu Pro Phe LeuPro Arg 1100 1105 1110 Ala Arg Ala Glu Arg Thr Val Met Arg Gln Asp AsnArg Asp Thr 1115 1120 1125 Val Asp Asp Thr Val Ser Ser Glu Ser Leu GlnSer Leu Phe Ser 1130 1135 1140 Glu Trp Asp Asn Pro Val Phe Ala Arg TyrPro Glu Val Ala Val 1145 1150 1155 Asp Val Ser Ser Gly Gln Ala Glu SerLeu Ala Val Lys Ile His 1160 1165 1170 Asn Ile Leu Tyr Pro Tyr Arg PheThr Lys Gly Met Ile His Ser 1175 1180 1185 Met Gln Val Leu Gln Gln ValAsp Asn Lys Phe Ile Ala Cys Leu 1190 1195 1200 Met Ser Thr Lys Thr GluGlu Asn Gly Glu Ala Asp Ser Tyr Glu 1205 1210 1215 Lys Gln Gln Ala GlnGly Ser Gly Arg Lys Lys Leu Leu Ser Ser 1220 1225 1230 Thr Leu Ile ProPro Leu Glu Ile Thr Val Thr Glu Glu Gln Arg 1235 1240 1245 Arg Leu LeuTrp Cys Tyr His Lys Asn Leu Glu Asp Leu Gly Leu 1250 1255 1260 Glu PheVal Phe Pro Asp Thr Ser Asp Ser Leu Val Leu Val Gly 1265 1270 1275 LysVal Pro Leu Cys Phe Val Glu Arg Glu Ala Asn Glu Leu Arg 1280 1285 1290Arg Gly Arg Ser Thr Val Thr Lys Ser Ile Val Glu Glu Phe Ile 1295 13001305 Arg Glu Gln Leu Glu Leu Leu Gln Thr Thr Gly Gly Ile Gln Gly 13101315 1320 Thr Leu Pro Leu Thr Val Gln Lys Val Leu Ala Ser Gln Ala Cys1325 1330 1335 His Gly Ala Ile Lys Phe Asn Asp Gly Leu Ser Leu Gln GluSer 1340 1345 1350 Cys Arg Leu Ile Glu Ala Leu Ser Ser Cys Gln Leu ProPhe Gln 1355 1360 1365 Cys Ala His Gly Arg Pro Ser Met Leu Pro Leu AlaAsp Ile Asp 1370 1375 1380 His Leu Glu Gln Glu Lys Gln Ile Lys Pro AsnLeu Thr Lys Leu 1385 1390 1395 Arg Lys Met Ala Gln Ala Trp Arg Leu PheGly Lys Ala Glu Cys 1400 1405 1410 Asp Thr Arg Gln Ser Leu Gln Gln SerMet Pro Pro Cys Glu Pro 1415 1420 1425 Pro 30 2340 DNA Arabidopsisthaliana 30 atgcaaggag attcttctcc gtctccgacg actactagct ctcctttgataagacctata 60 aacagaaacg taattcacag aatctgttcc ggtcaagtca tcttagacctctcttcggcc 120 gtcaaggagc ttgtcgagaa tagtctcgac gccggcgcca ccagtatagagattaacctc 180 cgagactacg gcgaagacta ttttcaggtc attgacaatg gttgtggcatttccccaacc 240 aatttcaagg tttgtgtcca aattctccga agaacttttg atgttcttgcacttaagcat 300 catacttcta aattagagga tttcacagat cttttgaatt tgactacttatggttttaga 360 ggagaagcct tgagctctct ctgtgcattg ggaaatctca ctgtggaaacaagaacaaag 420 aatgagccag ttgctacgct cttgacgttt gatcattctg gtttgcttactgctgaaaag 480 aagactgctc gccaaattgg taccactgtc actgttagga agttgttctctaatttacct 540 gtacgaagca aagagtttaa gcggaatata cgcaaagaat atgggaagcttgtatcttta 600 ttgaacgcat atgcgcttat tgcgaaagga gtgcggtttg tctgctctaacacgactggg 660 aaaaacccaa agtctgttgt gctgaacaca caagggaggg gttcacttaaagataatatc 720 ataacagttt tcggcattag tacctttaca agtctacagc ctggtactggacgcaattta 780 gcagatcgac agtatttctt tataaatggt cggcctgtag atatgccaaaagtcagcaag 840 ttggtgaatg agttatataa agatacaagt tctcggaaat atccagttaccattctggat 900 tttattgtgc ctggtggagc atgtgatttg aatgtcacgc ccgataaaagaaaggtgttc 960 ttttctgacg agacttctgt tatcggttct ttgagggaag gtctgaacgagatatattcc 1020 tccagtaatg cgtcttatat tgttaatagg ttcgaggaga attcggagcaaccagataag 1080 gctggagttt cgtcgtttca gaagaaatca aatcttttgt cagaagggatagttctggat 1140 gtcagttcta aaacaagact aggggaagct attgagaaag aaaatccatccttaagggag 1200 gttgaaattg ataatagttc gccaatggag aagtttaagt ttgagatcaaggcatgtggg 1260 acgaagaaag gggaaggttc tttatcagtc catgatgtaa ctcaccttgacaagacacct 1320 agcaaaggtt tgcctcagtt aaatgtgact gagaaagtta ctgatgcaagtaaagacttg 1380 agcagccgct ctagctttgc ccagtcaact ttgaatactt ttgttaccatgggaaaaaga 1440 aaacatgaaa acataagcac catcctctct gaaacacctg tcctcagaaaccaaacttct 1500 agttatcgtg tggagaaaag caaatttgaa gttcgtgcct tagcttcaaggtgtctcgtg 1560 gaaggcgatc aacttgatga tatggtcatc tcaaaggaag atatgacaccaagcgaaaga 1620 gattctgaac taggcaatcg gatttctcct ggaacacaag ctgataatgttgaaagacat 1680 gagagagtac tcgggcaatt caatcttggg ttcatcattg caaaattggagcgagatctg 1740 ttcattgtgg atcagcatgc agctgatgag aaattcaact tcgaacatttagcaaggtca 1800 actgtcctga accagcaacc cttactccag cctttgaact tggaactctctccagaagaa 1860 gaagtaactg tgttaatgca catggatatt atcagggaaa atggctttcttctagaggag 1920 aatccaagtg ctcctcccgg aaaacacttt agactacgag ccattccttatagcaagaat 1980 atcacctttg gagtcgaaga tcttaaagac ctgatctcaa ctctaggagataaccatggg 2040 gaatgttcgg ttgctagtag ctacaaaacc agcaaaacag attcgatttgtccatcacga 2100 gtccgtgcaa tgctagcatc ccgagcatgc agatcatctg tgatgatcggagatccactc 2160 agaaaaaacg aaatgcagaa gatagtagaa cacttggcag atctcgaatctccttggaat 2220 tgcccacacg gacgaccaac aatgcgtcat cttgtggact tgacaactttactcacatta 2280 cctgatgacg acaatgtcaa tgatgatgat gatgatgatg caaccatctcattggcatga 2340 31 779 PRT Arabidopsis thaliana 31 Met Gln Gly Asp SerSer Pro Ser Pro Thr Thr Thr Ser Ser Pro Leu 1 5 10 15 Ile Arg Pro IleAsn Arg Asn Val Ile His Arg Ile Cys Ser Gly Gln 20 25 30 Val Ile Leu AspLeu Ser Ser Ala Val Lys Glu Leu Val Glu Asn Ser 35 40 45 Leu Asp Ala GlyAla Thr Ser Ile Glu Ile Asn Leu Arg Asp Tyr Gly 50 55 60 Glu Asp Tyr PheGln Val Ile Asp Asn Gly Cys Gly Ile Ser Pro Thr 65 70 75 80 Asn Phe LysVal Cys Val Gln Ile Leu Arg Arg Thr Phe Asp Val Leu 85 90 95 Ala Leu LysHis His Thr Ser Lys Leu Glu Asp Phe Thr Asp Leu Leu 100 105 110 Asn LeuThr Thr Tyr Gly Phe Arg Gly Glu Ala Leu Ser Ser Leu Cys 115 120 125 AlaLeu Gly Asn Leu Thr Val Glu Thr Arg Thr Lys Asn Glu Pro Val 130 135 140Ala Thr Leu Leu Thr Phe Asp His Ser Gly Leu Leu Thr Ala Glu Lys 145 150155 160 Lys Thr Ala Arg Gln Ile Gly Thr Thr Val Thr Val Arg Lys Leu Phe165 170 175 Ser Asn Leu Pro Val Arg Ser Lys Glu Phe Lys Arg Asn Ile ArgLys 180 185 190 Glu Tyr Gly Lys Leu Val Ser Leu Leu Asn Ala Tyr Ala LeuIle Ala 195 200 205 Lys Gly Val Arg Phe Val Cys Ser Asn Thr Thr Gly LysAsn Pro Lys 210 215 220 Ser Val Val Leu Asn Thr Gln Gly Arg Gly Ser LeuLys Asp Asn Ile 225 230 235 240 Ile Thr Val Phe Gly Ile Ser Thr Phe ThrSer Leu Gln Pro Gly Thr 245 250 255 Gly Arg Asn Leu Ala Asp Arg Gln TyrPhe Phe Ile Asn Gly Arg Pro 260 265 270 Val Asp Met Pro Lys Val Ser LysLeu Val Asn Glu Leu Tyr Lys Asp 275 280 285 Thr Ser Ser Arg Lys Tyr ProVal Thr Ile Leu Asp Phe Ile Val Pro 290 295 300 Gly Gly Ala Cys Asp LeuAsn Val Thr Pro Asp Lys Arg Lys Val Phe 305 310 315 320 Phe Ser Asp GluThr Ser Val Ile Gly Ser Leu Arg Glu Gly Leu Asn 325 330 335 Glu Ile TyrSer Ser Ser Asn Ala Ser Tyr Ile Val Asn Arg Phe Glu 340 345 350 Glu AsnSer Glu Gln Pro Asp Lys Ala Gly Val Ser Ser Phe Gln Lys 355 360 365 LysSer Asn Leu Leu Ser Glu Gly Ile Val Leu Asp Val Ser Ser Lys 370 375 380Thr Arg Leu Gly Glu Ala Ile Glu Lys Glu Asn Pro Ser Leu Arg Glu 385 390395 400 Val Glu Ile Asp Asn Ser Ser Pro Met Glu Lys Phe Lys Phe Glu Ile405 410 415 Lys Ala Cys Gly Thr Lys Lys Gly Glu Gly Ser Leu Ser Val HisAsp 420 425 430 Val Thr His Leu Asp Lys Thr Pro Ser Lys Gly Leu Pro GlnLeu Asn 435 440 445 Val Thr Glu Lys Val Thr Asp Ala Ser Lys Asp Leu SerSer Arg Ser 450 455 460 Ser Phe Ala Gln Ser Thr Leu Asn Thr Phe Val ThrMet Gly Lys Arg 465 470 475 480 Lys His Glu Asn Ile Ser Thr Ile Leu SerGlu Thr Pro Val Leu Arg 485 490 495 Asn Gln Thr Ser Ser Tyr Arg Val GluLys Ser Lys Phe Glu Val Arg 500 505 510 Ala Leu Ala Ser Arg Cys Leu ValGlu Gly Asp Gln Leu Asp Asp Met 515 520 525 Val Ile Ser Lys Glu Asp MetThr Pro Ser Glu Arg Asp Ser Glu Leu 530 535 540 Gly Asn Arg Ile Ser ProGly Thr Gln Ala Asp Asn Val Glu Arg His 545 550 555 560 Glu Arg Val LeuGly Gln Phe Asn Leu Gly Phe Ile Ile Ala Lys Leu 565 570 575 Glu Arg AspLeu Phe Ile Val Asp Gln His Ala Ala Asp Glu Lys Phe 580 585 590 Asn PheGlu His Leu Ala Arg Ser Thr Val Leu Asn Gln Gln Pro Leu 595 600 605 LeuGln Pro Leu Asn Leu Glu Leu Ser Pro Glu Glu Glu Val Thr Val 610 615 620Leu Met His Met Asp Ile Ile Arg Glu Asn Gly Phe Leu Leu Glu Glu 625 630635 640 Asn Pro Ser Ala Pro Pro Gly Lys His Phe Arg Leu Arg Ala Ile Pro645 650 655 Tyr Ser Lys Asn Ile Thr Phe Gly Val Glu Asp Leu Lys Asp LeuIle 660 665 670 Ser Thr Leu Gly Asp Asn His Gly Glu Cys Ser Val Ala SerSer Tyr 675 680 685 Lys Thr Ser Lys Thr Asp Ser Ile Cys Pro Ser Arg ValArg Ala Met 690 695 700 Leu Ala Ser Arg Ala Cys Arg Ser Ser Val Met IleGly Asp Pro Leu 705 710 715 720 Arg Lys Asn Glu Met Gln Lys Ile Val GluHis Leu Ala Asp Leu Glu 725 730 735 Ser Pro Trp Asn Cys Pro His Gly ArgPro Thr Met Arg His Leu Val 740 745 750 Asp Leu Thr Thr Leu Leu Thr LeuPro Asp Asp Asp Asn Val Asn Asp 755 760 765 Asp Asp Asp Asp Asp Ala ThrIle Ser Leu Ala 770 775 32 3456 DNA Arabidopsis thaliana 32 atgaagacgatcaagccctt gccggaagga gttcgtcact ccatgcgttc tggaattatc 60 atgttcgacatggcgagggt cgtggaagaa ctcgtcttca acagtctcga tgctggggcg 120 accaaggtgtctatcttcgt gggtgttgtt tcatgctctg tgaaagttgt ggatgatgga 180 tcaggcgtttcaagagatga tttggttttg ttgggagaaa gatatgctac ttcaaagttt 240 cacgacttcaccaacgtgga gacagctagt gaaacttttg gatttcgtgg agaggcctta 300 gcttcaatatcagatatctc gttactggag gttaggacaa aagctattgg gaggcctaat 360 ggttatcgaaaggttatgaa gggatccaag tgtctacatc ttggaattga tgatgataga 420 aaagactctggcacgacggt aactgtccga gatctatttt acagtcagcc agtgagacga 480 aaatatatgcaatccagccc caagaaagtt ttggaatcta tcaaaaagtg tgtgttccgg 540 attgcccttgtgcactccaa tgtttccttc agtgttcttg atatcgaaag tgatgaagag 600 cttttccaaaccaatccttc ttcttcagca ttctcactac tgatgagaga tgcagggacc 660 gaagctgtaaattcgctttg taaagtaaac gttacagatg gcatgctgaa tgtctctggt 720 tttgagtgtgcggatgactg gaagcctacg gatgggcaac aaacaggaag acgcaataga 780 cttcaatccaaccctggtta cattctgtgc atagcatgtc cacgccgtct ttatgaattc 840 tcgtttgaaccatcaaagac gcacgttgag ttcaagaagt ggggacctgt acttgccttt 900 atagaaagaatcactctagc caactggaag aaagatagaa ttcttgaact ttttgatggg 960 ggagctgatatactggcaaa aggtgataga caagacctga ttgatgacaa aattagactt 1020 caaaacggcagccttttctc aattcttcat tttctggatg cagattggcc agaagctatg 1080 gaacctgcaaaaaagaagct gaagagaagt aatgatcatg caccttgtag ttctctcttg 1140 tttccgtctgctgactttaa acaagatggt gattattttt ctccacgaaa ggatgtatgg 1200 tctccagaatgtgaagtcga actgaaaatt cagaatccca aagagcaagg tactgtagct 1260 ggatttgaaagccggactga ttctcttcta cagtcacgtg acatagaaat gcaaacgaat 1320 gaagacttcccacaagttac tgacctcctt gaaacaagct tggttgctga ctctaagtgc 1380 cgtaaacagtttctaacaag atgtcagatt accacacctg tcaatatcaa ccatgatttt 1440 atgaaagattcagacgtgtt aaattttcag tttcaaggat tgaaagatga gttggatgtc 1500 agcaattgcattggaaagca tctcttgcgt ggttgctctt caagagtaag cctaaccttt 1560 catgagcctaaactatctca tgttgaaggg tatgaatccg tcgtgcctat gatacctaat 1620 gaaaaacaaagtagtccgcg ggtcctagag accagagaag gtggttcgta ctgtgatgtt 1680 tattctgataagactcctga ttgttcccta gggagttcat ggcaggatac tgattggttt 1740 actccacagtgttcctcaga taggggatgt gttggaattg gagaagattt taacattacc 1800 cccatagatactgcggaatt tgattcttat gatgaaaaag ttggtagtaa aaagtatctt 1860 tcttctgtcaatgtggggag ctctgttact ggtagtttct gtttaagttc tgagtggtct 1920 ccaatgtactccacaccttc tgcgaccaag tgggagtctg agtaccagaa aggttgtcga 1980 attcttgaacagagtttgag actgggaagg atgcctgacc ctgaattttg tttcagtgca 2040 gctaacaacatcaaatttga ccacgaggtc atacctgaaa tggattgctg tgaaaccggt 2100 acagactctttcacagctat tcagaactgc actcagttag ctgataaaat ttgcaagtct 2160 tcgtgggggcatgcagatga tgtgcgtatt gaccaatata gtatcaggaa ggaaaagttc 2220 agttatatggatggcacaca gaacaatgct ggtaaacaaa ggtcaaaaag aagtcgatct 2280 gctcctccattttatcgaga gaagaagaga tttatcagct taagttgtaa atcagacaca 2340 aaaccaaagaactctgatcc atcagaacct gatgatctgg agtgtttgac acaaccttgt 2400 aatgcatctcaaatgcatct taagtgcagc atccttgatg atgtgtcgta tgaccacata 2460 caagaaacagaaaaaagatt gagttctgcc tcagacttga aagcatctgc tggttgcagg 2520 actgtgcactcagagaccca agatgaggat gtgcacgaag acttcagctc agaggaattt 2580 ctggatccaattaaatccac aacaaaatgg cgccataact gtgcggtctc tcaggttccc 2640 aaggaatcacacgagcttca tggtcaagat ggtgtatttg atatatcttc gggacttctg 2700 cacttacgatccgatgaatc cttggttcct gaatctatca acagacactc ccttgaagat 2760 gccaaggttctacaacaggt tgataaaaaa tatatcccaa tcgttgcttg tggaacagtt 2820 gccatcgttgatcagcatgc tgccgatgaa agaattcgtt tggaagagct gcgtacaaag 2880 tttattaatgatgcattatt aatttttgtg ttgacattaa aggtactgcc ggagatgggt 2940 tatcagttactccagagtta ttcagagcag ataagagact ggggttggat ctgcaacatt 3000 actgtagaagggtcaacgtc ctttaagaaa aacatgagca tcatccagcg gaaaccaaca 3060 ccaatcacacttaatgcggt tccatgcatt ctgggtgtaa atctatcaga tgttgatcta 3120 ttagagtttcttcagcagct tgctgatact gacggatcat caactattcc tccatctgtt 3180 cttcgagtcctaaattccaa agcctgtaga ggtgcaatta tgtttggaga tagtctgtta 3240 ccgtcagaatgctctttaat cattgatgga ctgaagcaga cctcactttg tttccagtgt 3300 gctcatgggcgacctacaac agttcctctt gtcgatttga aggcattgca caaacagata 3360 gcaaagctcagtggaagaca agtgtggcat ggcttacaac gcagagaaat tacacttgat 3420 cgtgcaaaatcacgcttaga caacgctaaa agttaa 3456 33 1151 PRT Arabidopsis thaliana 33Met Lys Thr Ile Lys Pro Leu Pro Glu Gly Val Arg His Ser Met Arg 1 5 1015 Ser Gly Ile Ile Met Phe Asp Met Ala Arg Val Val Glu Glu Leu Val 20 2530 Phe Asn Ser Leu Asp Ala Gly Ala Thr Lys Val Ser Ile Phe Val Gly 35 4045 Val Val Ser Cys Ser Val Lys Val Val Asp Asp Gly Ser Gly Val Ser 50 5560 Arg Asp Asp Leu Val Leu Leu Gly Glu Arg Tyr Ala Thr Ser Lys Phe 65 7075 80 His Asp Phe Thr Asn Val Glu Thr Ala Ser Glu Thr Phe Gly Phe Arg 8590 95 Gly Glu Ala Leu Ala Ser Ile Ser Asp Ile Ser Leu Leu Glu Val Arg100 105 110 Thr Lys Ala Ile Gly Arg Pro Asn Gly Tyr Arg Lys Val Met LysGly 115 120 125 Ser Lys Cys Leu His Leu Gly Ile Asp Asp Asp Arg Lys AspSer Gly 130 135 140 Thr Thr Val Thr Val Arg Asp Leu Phe Tyr Ser Gln ProVal Arg Arg 145 150 155 160 Lys Tyr Met Gln Ser Ser Pro Lys Lys Val LeuGlu Ser Ile Lys Lys 165 170 175 Cys Val Phe Arg Ile Ala Leu Val His SerAsn Val Ser Phe Ser Val 180 185 190 Leu Asp Ile Glu Ser Asp Glu Glu LeuPhe Gln Thr Asn Pro Ser Ser 195 200 205 Ser Ala Phe Ser Leu Leu Met ArgAsp Ala Gly Thr Glu Ala Val Asn 210 215 220 Ser Leu Cys Lys Val Asn ValThr Asp Gly Met Leu Asn Val Ser Gly 225 230 235 240 Phe Glu Cys Ala AspAsp Trp Lys Pro Thr Asp Gly Gln Gln Thr Gly 245 250 255 Arg Arg Asn ArgLeu Gln Ser Asn Pro Gly Tyr Ile Leu Cys Ile Ala 260 265 270 Cys Pro ArgArg Leu Tyr Glu Phe Ser Phe Glu Pro Ser Lys Thr His 275 280 285 Val GluPhe Lys Lys Trp Gly Pro Val Leu Ala Phe Ile Glu Arg Ile 290 295 300 ThrLeu Ala Asn Trp Lys Lys Asp Arg Ile Leu Glu Leu Phe Asp Gly 305 310 315320 Gly Ala Asp Ile Leu Ala Lys Gly Asp Arg Gln Asp Leu Ile Asp Asp 325330 335 Lys Ile Arg Leu Gln Asn Gly Ser Leu Phe Ser Ile Leu His Phe Leu340 345 350 Asp Ala Asp Trp Pro Glu Ala Met Glu Pro Ala Lys Lys Lys LeuLys 355 360 365 Arg Ser Asn Asp His Ala Pro Cys Ser Ser Leu Leu Phe ProSer Ala 370 375 380 Asp Phe Lys Gln Asp Gly Asp Tyr Phe Ser Pro Arg LysAsp Val Trp 385 390 395 400 Ser Pro Glu Cys Glu Val Glu Leu Lys Ile GlnAsn Pro Lys Glu Gln 405 410 415 Gly Thr Val Ala Gly Phe Glu Ser Arg ThrAsp Ser Leu Leu Gln Ser 420 425 430 Arg Asp Ile Glu Met Gln Thr Asn GluAsp Phe Pro Gln Val Thr Asp 435 440 445 Leu Leu Glu Thr Ser Leu Val AlaAsp Ser Lys Cys Arg Lys Gln Phe 450 455 460 Leu Thr Arg Cys Gln Ile ThrThr Pro Val Asn Ile Asn His Asp Phe 465 470 475 480 Met Lys Asp Ser AspVal Leu Asn Phe Gln Phe Gln Gly Leu Lys Asp 485 490 495 Glu Leu Asp ValSer Asn Cys Ile Gly Lys His Leu Leu Arg Gly Cys 500 505 510 Ser Ser ArgVal Ser Leu Thr Phe His Glu Pro Lys Leu Ser His Val 515 520 525 Glu GlyTyr Glu Ser Val Val Pro Met Ile Pro Asn Glu Lys Gln Ser 530 535 540 SerPro Arg Val Leu Glu Thr Arg Glu Gly Gly Ser Tyr Cys Asp Val 545 550 555560 Tyr Ser Asp Lys Thr Pro Asp Cys Ser Leu Gly Ser Ser Trp Gln Asp 565570 575 Thr Asp Trp Phe Thr Pro Gln Cys Ser Ser Asp Arg Gly Cys Val Gly580 585 590 Ile Gly Glu Asp Phe Asn Ile Thr Pro Ile Asp Thr Ala Glu PheAsp 595 600 605 Ser Tyr Asp Glu Lys Val Gly Ser Lys Lys Tyr Leu Ser SerVal Asn 610 615 620 Val Gly Ser Ser Val Thr Gly Ser Phe Cys Leu Ser SerGlu Trp Ser 625 630 635 640 Pro Met Tyr Ser Thr Pro Ser Ala Thr Lys TrpGlu Ser Glu Tyr Gln 645 650 655 Lys Gly Cys Arg Ile Leu Glu Gln Ser LeuArg Leu Gly Arg Met Pro 660 665 670 Asp Pro Glu Phe Cys Phe Ser Ala AlaAsn Asn Ile Lys Phe Asp His 675 680 685 Glu Val Ile Pro Glu Met Asp CysCys Glu Thr Gly Thr Asp Ser Phe 690 695 700 Thr Ala Ile Gln Asn Cys ThrGln Leu Ala Asp Lys Ile Cys Lys Ser 705 710 715 720 Ser Trp Gly His AlaAsp Asp Val Arg Ile Asp Gln Tyr Ser Ile Arg 725 730 735 Lys Glu Lys PheSer Tyr Met Asp Gly Thr Gln Asn Asn Ala Gly Lys 740 745 750 Gln Arg SerLys Arg Ser Arg Ser Ala Pro Pro Phe Tyr Arg Glu Lys 755 760 765 Lys ArgPhe Ile Ser Leu Ser Cys Lys Ser Asp Thr Lys Pro Lys Asn 770 775 780 SerAsp Pro Ser Glu Pro Asp Asp Leu Glu Cys Leu Thr Gln Pro Cys 785 790 795800 Asn Ala Ser Gln Met His Leu Lys Cys Ser Ile Leu Asp Asp Val Ser 805810 815 Tyr Asp His Ile Gln Glu Thr Glu Lys Arg Leu Ser Ser Ala Ser Asp820 825 830 Leu Lys Ala Ser Ala Gly Cys Arg Thr Val His Ser Glu Thr GlnAsp 835 840 845 Glu Asp Val His Glu Asp Phe Ser Ser Glu Glu Phe Leu AspPro Ile 850 855 860 Lys Ser Thr Thr Lys Trp Arg His Asn Cys Ala Val SerGln Val Pro 865 870 875 880 Lys Glu Ser His Glu Leu His Gly Gln Asp GlyVal Phe Asp Ile Ser 885 890 895 Ser Gly Leu Leu His Leu Arg Ser Asp GluSer Leu Val Pro Glu Ser 900 905 910 Ile Asn Arg His Ser Leu Glu Asp AlaLys Val Leu Gln Gln Val Asp 915 920 925 Lys Lys Tyr Ile Pro Ile Val AlaCys Gly Thr Val Ala Ile Val Asp 930 935 940 Gln His Ala Ala Asp Glu ArgIle Arg Leu Glu Glu Leu Arg Thr Lys 945 950 955 960 Phe Ile Asn Asp AlaLeu Leu Ile Phe Val Leu Thr Leu Lys Val Leu 965 970 975 Pro Glu Met GlyTyr Gln Leu Leu Gln Ser Tyr Ser Glu Gln Ile Arg 980 985 990 Asp Trp GlyTrp Ile Cys Asn Ile Thr Val Glu Gly Ser Thr Ser Phe 995 1000 1005 LysLys Asn Met Ser Ile Ile Gln Arg Lys Pro Thr Pro Ile Thr 1010 1015 1020Leu Asn Ala Val Pro Cys Ile Leu Gly Val Asn Leu Ser Asp Val 1025 10301035 Asp Leu Leu Glu Phe Leu Gln Gln Leu Ala Asp Thr Asp Gly Ser 10401045 1050 Ser Thr Ile Pro Pro Ser Val Leu Arg Val Leu Asn Ser Lys Ala1055 1060 1065 Cys Arg Gly Ala Ile Met Phe Gly Asp Ser Leu Leu Pro SerGlu 1070 1075 1080 Cys Ser Leu Ile Ile Asp Gly Leu Lys Gln Thr Ser LeuCys Phe 1085 1090 1095 Gln Cys Ala His Gly Arg Pro Thr Thr Val Pro LeuVal Asp Leu 1100 1105 1110 Lys Ala Leu His Lys Gln Ile Ala Lys Leu SerGly Arg Gln Val 1115 1120 1125 Trp His Gly Leu Gln Arg Arg Glu Ile ThrLeu Asp Arg Ala Lys 1130 1135 1140 Ser Arg Leu Asp Asn Ala Lys Ser 11451150 34 3330 DNA Arabidopsis thaliana 34 atgcagcgcc agagatcgattttgtctttc ttccaaaaac ccacggcggc gactacgaag 60 ggtttggttt ccggcgatgctgctagcggc gggggcggca gcggaggacc acgatttaat 120 gtgaaggaag gggatgctaaaggcgacgct tctgtacgtt ttgctgtttc gaaatctgtc 180 gatgaggtta gaggaacggatactccaccg gagaaggttc cgcgtcgtgt cctgccgtct 240 ggatttaagc cggctgaatccgccggtgat gcttcgtccc tgttctccaa tattatgcat 300 aagtttgtaa aagtcgatgatcgagattgt tctggagaga ggagccgaga agatgttgtt 360 ccgctgaatg attcatctctatgtatgaag gctaatgatg ttattcctca atttcgttcc 420 aataatggta aaactcaagaaagaaaccat gcttttagtt tcagtgggag agctgaactt 480 agatcagtag aagatataggagtagatggc gatgttcctg gtccagaaac accagggatg 540 cgtccacgtg cttctcgcttgaagcgagtt ctggaggatg aaatgacttt taaggaggat 600 aaggttcctg tattggactctaacaaaagg ctgaaaatgc tccaggatcc ggtttgtgga 660 gagaagaaag aagtaaacgaaggaaccaaa tttgaatggc ttgagtcttc tcgaatcagg 720 gatgccaata gaagacgtcctgatgatccc ctttacgata gaaagacctt acacatacca 780 cctgatgttt tcaagaaaatgtctgcatca caaaagcaat attggagtgt taagagtgaa 840 tatatggaca ttgtgcttttctttaaagtg gggaaatttt atgagctgta tgagctagat 900 gcggaattag gtcacaaggagcttgactgg aagatgacca tgagtggtgt gggaaaatgc 960 agacaggttg gtatctctgaaagtgggata gatgaggcag tgcaaaagct attagctcgt 1020 ggatataaag ttggacgaatcgagcagcta gaaacatctg accaagcaaa agccagaggt 1080 gctaatacta taattccaaggaagctagtt caggtattaa ctccatcaac agcaagcgag 1140 ggaaacatcg ggcctgatgccgtccatctt cttgctataa aagagatcaa aatggagcta 1200 caaaagtgtt caactgtgtatggatttgct tttgttgact gtgctgcctt gaggttttgg 1260 gttgggtcca tcagcgatgatgcatcatgt gctgctcttg gagcgttatt gatgcaggtt 1320 tctccaaagg aagtgttatatgacagtaaa gggctatcaa gagaagcaca aaaggctcta 1380 aggaaatata cgttgacagggtctacggcg gtacagttgg ctccagtacc acaagtaatg 1440 ggggatacag atgctgctggagttagaaat ataatagaat ctaacggata ctttaaaggt 1500 tcttctgaat catggaactgtgctgttgat ggtctaaatg aatgtgatgt tgcccttagt 1560 gctcttggag agctaattaatcatctgtct aggctaaagc tagaagatgt acttaagcat 1620 ggggatattt ttccataccaagtttacagg ggttgtctca gaattgatgg ccagacgatg 1680 gtaaatcttg agatatttaacaatagctgt gatggtggtc cttcagggac cttgtacaaa 1740 tatcttgata actgtgttagtccaactggt aagcgactct taaggaattg gatctgccat 1800 ccactcaaag atgtagaaagcatcaataaa cggcttgatg tagttgaaga attcacggca 1860 aactcagaaa gtatgcaaatcactggccag tatctccaca aacttccaga cttagaaaga 1920 ctgctcggac gcatcaagtctagcgttcga tcatcagcct ctgtgttgcc tgctcttctg 1980 gggaaaaaag tgctgaaacaacgagttaaa gcatttgggc aaattgtgaa agggttcaga 2040 agtggaattg atctgttgttggctctacag aaggaatcaa atatgatgag tttgctttat 2100 aaactctgta aacttcctatattagtagga aaaagcgggc tagagttatt tctttctcaa 2160 ttcgaagcag ccatagatagcgactttcca aattatcaga accaagatgt gacagatgaa 2220 aacgctgaaa ctctcacaatacttatcgaa ctttttatcg aaagagcaac tcaatggtct 2280 gaggtcattc acaccataagctgcctagat gtcctgagat cttttgcaat cgcagcaagt 2340 ctctctgctg gaagcatggccaggcctgtt atttttcccg aatcagaagc tacagatcag 2400 aatcagaaaa caaaagggccaatacttaaa atccaaggac tatggcatcc atttgcagtt 2460 gcagccgatg gtcaattgcctgttccgaat gatatactcc ttggcgaggc tagaagaagc 2520 agtggcagca ttcatcctcggtcattgtta ctgacgggac caaacatggg cggaaaatca 2580 actcttcttc gtgcaacatgtctggccgtt atctttgccc aacttggctg ctacgtgccg 2640 tgtgagtctt gcgaaatctccctcgtggat actatcttca caaggcttgg cgcatctgat 2700 agaatcatga caggagagagtacctttttg gtagaatgca ctgagacagc gtcagttctt 2760 cagaatgcaa ctcaggattcactagtaatc cttgacgaac tgggcagagg aactagtact 2820 ttcgatggat acgccattgcatactcggtt tttcgtcacc tggtagagaa agttcaatgt 2880 cggatgctct ttgcaacacattaccaccct ctcaccaagg aattcgcgtc tcacccacgt 2940 gtcacctcga aacacatggcttgcgcattc aaatcaagat ctgattatca accacgtggt 3000 tgtgatcaag acctagtgttcttgtaccgt ttaaccgagg gagcttgtcc tgagagctac 3060 ggacttcaag tggcactcatggctggaata ccaaaccaag tggttgaaac agcatcaggt 3120 gctgctcaag ccatgaagagatcaattggg gaaaacttca agtcaagtga gctaagatct 3180 gagttctcaa gtctgcatgaagactggctc aagtcattgg tgggtatttc tcgagtcgcc 3240 cacaacaatg cccccattggcgaagatgac tacgacactt tgttttgctt atggcatgag 3300 atcaaatcct cttactgtgttcccaaataa 3330 35 1109 PRT Arabidopsis thaliana 35 Met Gln Arg Gln ArgSer Ile Leu Ser Phe Phe Gln Lys Pro Thr Ala 1 5 10 15 Ala Thr Thr LysGly Leu Val Ser Gly Asp Ala Ala Ser Gly Gly Gly 20 25 30 Gly Ser Gly GlyPro Arg Phe Asn Val Lys Glu Gly Asp Ala Lys Gly 35 40 45 Asp Ala Ser ValArg Phe Ala Val Ser Lys Ser Val Asp Glu Val Arg 50 55 60 Gly Thr Asp ThrPro Pro Glu Lys Val Pro Arg Arg Val Leu Pro Ser 65 70 75 80 Gly Phe LysPro Ala Glu Ser Ala Gly Asp Ala Ser Ser Leu Phe Ser 85 90 95 Asn Ile MetHis Lys Phe Val Lys Val Asp Asp Arg Asp Cys Ser Gly 100 105 110 Glu ArgSer Arg Glu Asp Val Val Pro Leu Asn Asp Ser Ser Leu Cys 115 120 125 MetLys Ala Asn Asp Val Ile Pro Gln Phe Arg Ser Asn Asn Gly Lys 130 135 140Thr Gln Glu Arg Asn His Ala Phe Ser Phe Ser Gly Arg Ala Glu Leu 145 150155 160 Arg Ser Val Glu Asp Ile Gly Val Asp Gly Asp Val Pro Gly Pro Glu165 170 175 Thr Pro Gly Met Arg Pro Arg Ala Ser Arg Leu Lys Arg Val LeuGlu 180 185 190 Asp Glu Met Thr Phe Lys Glu Asp Lys Val Pro Val Leu AspSer Asn 195 200 205 Lys Arg Leu Lys Met Leu Gln Asp Pro Val Cys Gly GluLys Lys Glu 210 215 220 Val Asn Glu Gly Thr Lys Phe Glu Trp Leu Glu SerSer Arg Ile Arg 225 230 235 240 Asp Ala Asn Arg Arg Arg Pro Asp Asp ProLeu Tyr Asp Arg Lys Thr 245 250 255 Leu His Ile Pro Pro Asp Val Phe LysLys Met Ser Ala Ser Gln Lys 260 265 270 Gln Tyr Trp Ser Val Lys Ser GluTyr Met Asp Ile Val Leu Phe Phe 275 280 285 Lys Val Gly Lys Phe Tyr GluLeu Tyr Glu Leu Asp Ala Glu Leu Gly 290 295 300 His Lys Glu Leu Asp TrpLys Met Thr Met Ser Gly Val Gly Lys Cys 305 310 315 320 Arg Gln Val GlyIle Ser Glu Ser Gly Ile Asp Glu Ala Val Gln Lys 325 330 335 Leu Leu AlaArg Gly Tyr Lys Val Gly Arg Ile Glu Gln Leu Glu Thr 340 345 350 Ser AspGln Ala Lys Ala Arg Gly Ala Asn Thr Ile Ile Pro Arg Lys 355 360 365 LeuVal Gln Val Leu Thr Pro Ser Thr Ala Ser Glu Gly Asn Ile Gly 370 375 380Pro Asp Ala Val His Leu Leu Ala Ile Lys Glu Ile Lys Met Glu Leu 385 390395 400 Gln Lys Cys Ser Thr Val Tyr Gly Phe Ala Phe Val Asp Cys Ala Ala405 410 415 Leu Arg Phe Trp Val Gly Ser Ile Ser Asp Asp Ala Ser Cys AlaAla 420 425 430 Leu Gly Ala Leu Leu Met Gln Val Ser Pro Lys Glu Val LeuTyr Asp 435 440 445 Ser Lys Gly Leu Ser Arg Glu Ala Gln Lys Ala Leu ArgLys Tyr Thr 450 455 460 Leu Thr Gly Ser Thr Ala Val Gln Leu Ala Pro ValPro Gln Val Met 465 470 475 480 Gly Asp Thr Asp Ala Ala Gly Val Arg AsnIle Ile Glu Ser Asn Gly 485 490 495 Tyr Phe Lys Gly Ser Ser Glu Ser TrpAsn Cys Ala Val Asp Gly Leu 500 505 510 Asn Glu Cys Asp Val Ala Leu SerAla Leu Gly Glu Leu Ile Asn His 515 520 525 Leu Ser Arg Leu Lys Leu GluAsp Val Leu Lys His Gly Asp Ile Phe 530 535 540 Pro Tyr Gln Val Tyr ArgGly Cys Leu Arg Ile Asp Gly Gln Thr Met 545 550 555 560 Val Asn Leu GluIle Phe Asn Asn Ser Cys Asp Gly Gly Pro Ser Gly 565 570 575 Thr Leu TyrLys Tyr Leu Asp Asn Cys Val Ser Pro Thr Gly Lys Arg 580 585 590 Leu LeuArg Asn Trp Ile Cys His Pro Leu Lys Asp Val Glu Ser Ile 595 600 605 AsnLys Arg Leu Asp Val Val Glu Glu Phe Thr Ala Asn Ser Glu Ser 610 615 620Met Gln Ile Thr Gly Gln Tyr Leu His Lys Leu Pro Asp Leu Glu Arg 625 630635 640 Leu Leu Gly Arg Ile Lys Ser Ser Val Arg Ser Ser Ala Ser Val Leu645 650 655 Pro Ala Leu Leu Gly Lys Lys Val Leu Lys Gln Arg Val Lys AlaPhe 660 665 670 Gly Gln Ile Val Lys Gly Phe Arg Ser Gly Ile Asp Leu LeuLeu Ala 675 680 685 Leu Gln Lys Glu Ser Asn Met Met Ser Leu Leu Tyr LysLeu Cys Lys 690 695 700 Leu Pro Ile Leu Val Gly Lys Ser Gly Leu Glu LeuPhe Leu Ser Gln 705 710 715 720 Phe Glu Ala Ala Ile Asp Ser Asp Phe ProAsn Tyr Gln Asn Gln Asp 725 730 735 Val Thr Asp Glu Asn Ala Glu Thr LeuThr Ile Leu Ile Glu Leu Phe 740 745 750 Ile Glu Arg Ala Thr Gln Trp SerGlu Val Ile His Thr Ile Ser Cys 755 760 765 Leu Asp Val Leu Arg Ser PheAla Ile Ala Ala Ser Leu Ser Ala Gly 770 775 780 Ser Met Ala Arg Pro ValIle Phe Pro Glu Ser Glu Ala Thr Asp Gln 785 790 795 800 Asn Gln Lys ThrLys Gly Pro Ile Leu Lys Ile Gln Gly Leu Trp His 805 810 815 Pro Phe AlaVal Ala Ala Asp Gly Gln Leu Pro Val Pro Asn Asp Ile 820 825 830 Leu LeuGly Glu Ala Arg Arg Ser Ser Gly Ser Ile His Pro Arg Ser 835 840 845 LeuLeu Leu Thr Gly Pro Asn Met Gly Gly Lys Ser Thr Leu Leu Arg 850 855 860Ala Thr Cys Leu Ala Val Ile Phe Ala Gln Leu Gly Cys Tyr Val Pro 865 870875 880 Cys Glu Ser Cys Glu Ile Ser Leu Val Asp Thr Ile Phe Thr Arg Leu885 890 895 Gly Ala Ser Asp Arg Ile Met Thr Gly Glu Ser Thr Phe Leu ValGlu 900 905 910 Cys Thr Glu Thr Ala Ser Val Leu Gln Asn Ala Thr Gln AspSer Leu 915 920 925 Val Ile Leu Asp Glu Leu Gly Arg Gly Thr Ser Thr PheAsp Gly Tyr 930 935 940 Ala Ile Ala Tyr Ser Val Phe Arg His Leu Val GluLys Val Gln Cys 945 950 955 960 Arg Met Leu Phe Ala Thr His Tyr His ProLeu Thr Lys Glu Phe Ala 965 970 975 Ser His Pro Arg Val Thr Ser Lys HisMet Ala Cys Ala Phe Lys Ser 980 985 990 Arg Ser Asp Tyr Gln Pro Arg GlyCys Asp Gln Asp Leu Val Phe Leu 995 1000 1005 Tyr Arg Leu Thr Glu GlyAla Cys Pro Glu Ser Tyr Gly Leu Gln 1010 1015 1020 Val Ala Leu Met AlaGly Ile Pro Asn Gln Val Val Glu Thr Ala 1025 1030 1035 Ser Gly Ala AlaGln Ala Met Lys Arg Ser Ile Gly Glu Asn Phe 1040 1045 1050 Lys Ser SerGlu Leu Arg Ser Glu Phe Ser Ser Leu His Glu Asp 1055 1060 1065 Trp LeuLys Ser Leu Val Gly Ile Ser Arg Val Ala His Asn Asn 1070 1075 1080 AlaPro Ile Gly Glu Asp Asp Tyr Asp Thr Leu Phe Cys Leu Trp 1085 1090 1095His Glu Ile Lys Ser Ser Tyr Cys Val Pro Lys 1100 1105 36 1170 DNA Homosapiens 36 atggcgcaac caaagcaaga gagggtggcg cgtgccagac accaacggtcggaaaccgcc 60 agacaccaac ggtcggaaac cgccaagaca ccaacgctcg gaaaccgccagacaccaacg 120 ctcggaaacc gccagacacc aaggctcgga atccacgcca ggccacgacggagggcgact 180 acctcccttc tgaccctgct gctggcgttc ggaaaaaacg cagtccggtgtgctctgatt 240 ggtccaggct ctttgacgtc acggactcga cctttgacag agccactaggcgaaaaggag 300 agacgggaag tattttttcc gccccgcccg gaaagggtgg agcacaacgtcgaaagcagc 360 cgttgggagc ccaggaggcg gggcgcctgt gggagccgtg gagggaactttcccagtccc 420 cgaggcggat ccggtgttgc atccttggag cgagctgaga actcgagtacagaacctgct 480 aaggccatca aacctattga tcggaagtca gtccatcaga tttgctctgggccggtggta 540 ccgagtctaa ggccgaatgc ggtgaaggag ttagtagaaa acagtctggatgctggtgcc 600 actaatgttg atctaaagct taaggactat ggagtggatc tcattgaagtttcaggcaat 660 ggatgtgggg tagaagaaga aaacttcgaa ggctttactc tgaaacatcacacatgtaag 720 attcaagagt ttgccgacct aactcaggtg gaaacttttg gctttcggggggaagctctg 780 agctcacttt gtgcactgag tgatgtcacc atttctacct gccgtgtatcagcgaaggtt 840 gggactcgac tggtgtttga tcactatggg aaaatcatcc agaaaaccccctacccccgc 900 cccagaggga tgacagtcag cgtgaagcag ttattttcta cgctacctgtgcaccataaa 960 gaatttcaaa ggaatattaa gaagaaacgt gcctgcttcc ccttcgccttctgccgtgat 1020 tgtcagtttc ctgaggcctc cccagccatg cttcctgtac agcctgtagaactgactcct 1080 agaagtaccc caccccaccc ctgctccttg gaggacaacg tgatcactgtattcagctct 1140 gtcaagaatg gtccaggttc ttctagatga 1170 37 389 PRT Homosapiens 37 Met Ala Gln Pro Lys Gln Glu Arg Val Ala Arg Ala Arg His GlnArg 1 5 10 15 Ser Glu Thr Ala Arg His Gln Arg Ser Glu Thr Ala Lys ThrPro Thr 20 25 30 Leu Gly Asn Arg Gln Thr Pro Thr Leu Gly Asn Arg Gln ThrPro Arg 35 40 45 Leu Gly Ile His Ala Arg Pro Arg Arg Arg Ala Thr Thr SerLeu Leu 50 55 60 Thr Leu Leu Leu Ala Phe Gly Lys Asn Ala Val Arg Cys AlaLeu Ile 65 70 75 80 Gly Pro Gly Ser Leu Thr Ser Arg Thr Arg Pro Leu ThrGlu Pro Leu 85 90 95 Gly Glu Lys Glu Arg Arg Glu Val Phe Phe Pro Pro ArgPro Glu Arg 100 105 110 Val Glu His Asn Val Glu Ser Ser Arg Trp Glu ProArg Arg Arg Gly 115 120 125 Ala Cys Gly Ser Arg Gly Gly Asn Phe Pro SerPro Arg Gly Gly Ser 130 135 140 Gly Val Ala Ser Leu Glu Arg Ala Glu AsnSer Ser Thr Glu Pro Ala 145 150 155 160 Lys Ala Ile Lys Pro Ile Asp ArgLys Ser Val His Gln Ile Cys Ser 165 170 175 Gly Pro Val Val Pro Ser LeuArg Pro Asn Ala Val Lys Glu Leu Val 180 185 190 Glu Asn Ser Leu Asp AlaGly Ala Thr Asn Val Asp Leu Lys Leu Lys 195 200 205 Asp Tyr Gly Val AspLeu Ile Glu Val Ser Gly Asn Gly Cys Gly Val 210 215 220 Glu Glu Glu AsnPhe Glu Gly Phe Thr Leu Lys His His Thr Cys Lys 225 230 235 240 Ile GlnGlu Phe Ala Asp Leu Thr Gln Val Glu Thr Phe Gly Phe Arg 245 250 255 GlyGlu Ala Leu Ser Ser Leu Cys Ala Leu Ser Asp Val Thr Ile Ser 260 265 270Thr Cys Arg Val Ser Ala Lys Val Gly Thr Arg Leu Val Phe Asp His 275 280285 Tyr Gly Lys Ile Ile Gln Lys Thr Pro Tyr Pro Arg Pro Arg Gly Met 290295 300 Thr Val Ser Val Lys Gln Leu Phe Ser Thr Leu Pro Val His His Lys305 310 315 320 Glu Phe Gln Arg Asn Ile Lys Lys Lys Arg Ala Cys Phe ProPhe Ala 325 330 335 Phe Cys Arg Asp Cys Gln Phe Pro Glu Ala Ser Pro AlaMet Leu Pro 340 345 350 Val Gln Pro Val Glu Leu Thr Pro Arg Ser Thr ProPro His Pro Cys 355 360 365 Ser Leu Glu Asp Asn Val Ile Thr Val Phe SerSer Val Lys Asn Gly 370 375 380 Pro Gly Ser Ser Arg 385 38 795 DNA Homosapiens 38 atgtgtcctt ggcggcctag actaggccgt cgctgtatgg tgagccccagggaggcggat 60 ctgggccccc agaaggacac ccgcctggat ttgccccgta gcccggcccgggcccctcgg 120 gagcagaaca gccttggtga ggtggacagg aggggacctc gcgagcagacgcgcgcgcca 180 gcgacagcag ccccgccccg gcctctcggg agccgggggg cagaggctgcggagccccag 240 gagggtctat cagccacagt ctctgcatgt ttccaagagc aacaggaaatgaacacattg 300 caggggccag tgtcattcaa agatgtggct gtggatttca cccaggaggagtggcggcaa 360 ctggaccctg atgagaagat agcatacggg gatgtgatgt tggagaactacagccatcta 420 gtttctgtgg ggtatgatta tcaccaagcc aaacatcatc atggagtggaggtgaaggaa 480 gtggagcagg gagaggagcc gtggataatg gaaggtgaat ttccatgtcaacatagtcca 540 gaacctgcta aggccatcaa acctattgat cggaagtcag tccatcagatttgctctggg 600 ccagtggtac tgagtctaag cactgcagtg aaggagttag tagaaaacagtctggatgct 660 ggtgccacta atattgatct aaagcttaag gactatggag tggatctcattgaagtttca 720 gacaatggat gtggggtaga agaagaaaac tttgaaggct taatctctttcagctctgaa 780 acatcacaca tgtaa 795 39 264 PRT Homo sapiens 39 Met CysPro Trp Arg Pro Arg Leu Gly Arg Arg Cys Met Val Ser Pro 1 5 10 15 ArgGlu Ala Asp Leu Gly Pro Gln Lys Asp Thr Arg Leu Asp Leu Pro 20 25 30 ArgSer Pro Ala Arg Ala Pro Arg Glu Gln Asn Ser Leu Gly Glu Val 35 40 45 AspArg Arg Gly Pro Arg Glu Gln Thr Arg Ala Pro Ala Thr Ala Ala 50 55 60 ProPro Arg Pro Leu Gly Ser Arg Gly Ala Glu Ala Ala Glu Pro Gln 65 70 75 80Glu Gly Leu Ser Ala Thr Val Ser Ala Cys Phe Gln Glu Gln Gln Glu 85 90 95Met Asn Thr Leu Gln Gly Pro Val Ser Phe Lys Asp Val Ala Val Asp 100 105110 Phe Thr Gln Glu Glu Trp Arg Gln Leu Asp Pro Asp Glu Lys Ile Ala 115120 125 Tyr Gly Asp Val Met Leu Glu Asn Tyr Ser His Leu Val Ser Val Gly130 135 140 Tyr Asp Tyr His Gln Ala Lys His His His Gly Val Glu Val LysGlu 145 150 155 160 Val Glu Gln Gly Glu Glu Pro Trp Ile Met Glu Gly GluPhe Pro Cys 165 170 175 Gln His Ser Pro Glu Pro Ala Lys Ala Ile Lys ProIle Asp Arg Lys 180 185 190 Ser Val His Gln Ile Cys Ser Gly Pro Val ValLeu Ser Leu Ser Thr 195 200 205 Ala Val Lys Glu Leu Val Glu Asn Ser LeuAsp Ala Gly Ala Thr Asn 210 215 220 Ile Asp Leu Lys Leu Lys Asp Tyr GlyVal Asp Leu Ile Glu Val Ser 225 230 235 240 Asp Asn Gly Cys Gly Val GluGlu Glu Asn Phe Glu Gly Leu Ile Ser 245 250 255 Phe Ser Ser Glu Thr SerHis Met 260

What is claimed is:
 1. A method for generating antibiotic resistantbacteria comprising the steps of: blocking mismatch repair in abacterium whereby said bacterium becomes hypermutable; contacting saidbacterium with at least one antibiotic; selecting said a bacterium thatis resistant to said antibiotic; and culturing said bacterium; therebygenerating antibiotic resistant bacteria.
 2. The method of claim 1wherein said mismatch repair is blocked by introducing a dominantnegative allele of a mismatch repair gene into said bacterium.
 3. Themethod of claim 2 wherein said dominant negative allele of a mismatchrepair gene is a PMS2-134 gene.
 4. The method of claim 1 wherein saidmismatch repair is blocked by introducing an antisense nucleic acidmolecule into said bacterium wherein said antisense nucleic acidmolecule specifically binds to a mismatch repair gene and inhibitsmismatch repair in said bacterium.
 5. The method of claim 1 wherein saidmismatch repair is blocked by exposing said bacterium to a compound thatinhibits mismatch repair.
 6. The method of claim 5 wherein said compoundis an anthracene derivative having the formula: wherein R₁-R₁₀ areindependently hydrogen, hydroxyl, amino, alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, O-alkyl,S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, O-alkynyl, S-alkynyl,N-alkynyl, aryl, substituted aryl, aryloxy, substituted aryloxy,heteroaryl, substituted heteroaryl, aralkyloxy, arylalkyl, alkylaryl,alkylaryloxy, arylsulfonyl, alkylsulfonyl, alkoxycarbonyl,aryloxycarbonyl, guanidino, carboxy, an alcohol, an amino acid,sulfonate, alkyl sulfonate, CN, NO₂, an aldehyde group, an ester, anether, a crown ether, a ketone, an organosulfur compound, anorganometallic group, a carboxylic acid, an organosilicon or acarbohydrate that optionally contains one or more alkylated hydroxylgroups; wherein said heteroalkyl, heteroaryl, and substituted heteroarylcontain at least one heteroatom that is oxygen, sulfur, a metal atom,phosphorus, silicon or nitrogen; wherein said substituents of saidsubstituted alkyl, substituted alkenyl, substituted alkynyl, substitutedaryl, and substituted heteroaryl are halogen, CN, NO₂, lower alkyl,aryl, heteroaryl, aralkyl, aralkyloxy, guanidino, alkoxycarbonyl,alkoxy, hydroxy, carboxy and amino; and wherein said amino groupsoptionally substituted with an acyl group, or 1 to 3 aryl or lower alkylgroups.
 7. The method of claim 6 wherein said compound is selected fromthe group consisting of 1,2-dimethylanthracene, 9,10-dimethylanthracene, 7,8-dimethylanthracene, 9,10-diphenylanthracene,9,10-dihydroxymethylanthracene, 9-hydroxymethyl-10-methylanthracene,dimethylanthracene-1,2-diol,9-hydroxymethyl-10-methylanthracene-1,2-diol,9-hydroxymethyl-10-methylanthracene-3,4-diol, 9,10-di-m-tolyanthracene.8. The method of claim 6, further comprising exposing said bacterium toa chemical mutagen.
 9. The method of claim 8 wherein said chemicalmutagen is selected from the group consisting of methane sulfonate,dimethyl sulfonate, O-6-methyl benzadine, ethylnitrosourea, ethidiumbromide, ethyl methanesulfonate, N-methyl-N′-nitro-N-nitrosoguanidine,methylnitrosourea, Tamoxifen, and 8-hydroxyguanine.
 10. The method ofclaim 5 wherein said compound is selected from the group consisting ofan ATP analog, a nuclease inhibitor, and a DNA polymerase inhibitor. 11.The method of claim 10 wherein said ATP analog is selected from thegroup consisting of AMP-PNP and ATP[gamma]S.
 12. The method of claim 10wherein said nuclease inhibitor is selected from the group consisting ofN-ethylmaleimide, heterodimeric adenine-chain-acridine compounds,exonulcease III inhibitors and heliquinomycin.
 13. The method of claim10 wherein said DNA polymerase inhibitor is selected from the groupconsisting of actinomycin D analogs, aphidicolin,1-(2′-Deoxy-2′-fluoro-beta-L-arabinofuranosyl)-5-methyluracil, and2′,3′-dideoxyribonucleoside 5′-triphosphates.
 14. The method of claim 1wherein said antibiotic is a quinilone.
 15. The method of claim 1wherein said antibiotic is an aminoglycoside.
 16. The method of claim 1wherein said antibiotic is a magainin.
 17. The method of claim 1 whereinsaid antibiotic is a defensin.
 18. The method of claim 1 wherein saidantibiotic is a tetracycline.
 19. The method of claim 1 wherein saidantibiotic is a beta-lactam.
 20. The method of claim 1 wherein saidantibiotic is a macrolide.
 21. The method of claim 1 wherein saidantibiotic is a lincosamide.
 22. The method of claim 1 wherein saidantibiotic is a sulfonamide.
 23. The method of claim 1 wherein saidantibiotic is a chloramphenicol.
 24. The method of claim 1 wherein saidantibiotic is a nitrofurantoin.
 25. The method of claim 1 wherein saidantibiotic is an isoniazid.
 26. The method of claim 1 wherein the stepof determining whether said bacterium is resistant to said antibioticcomprises analyzing said bacterium for multiantiboitic resistance. 27.The method of claim 1 further comprising making antibiotic resistantbacteria genetically stable.
 28. The method of claim 5 furthercomprising making antibiotic resistant bacteria genetically stable. 29.The method of claim 28 wherein said antibiotic resistant bacteria aremade genetically stable by removing the MMR inhibitory molecule.
 30. Amethod for identifying a mutant gene conferring antibiotic resistancecomprising comparing the genome of antibiotic resistant bacterium madeby the method of claim 1 to the genome of a wild-type strain of saidbacterium.
 31. The method of claim 30 wherein the genome of saidantibiotic resistant bacterium and the genome of said wild-type strainof said bacterium are compared by sequence analysis of the entiregenomes.
 32. The method of claim 30 wherein the genome of saidantibiotic resistant bacterium and the genome of said wild-type strainof said bacterium are compared by microarray analysis.
 33. The method ofclaim 30 wherein the genome of said antibiotic resistant bacterium andthe genome of said wild-type strain of said bacterium are compared by:introducing gene fragments from said antibiotic resistant bacterium intothe wild-type bacterium, thereby producing mutant bacteria; selecting amutant bacterium with antibiotic resistance; and sequencing said genefragment from said mutant bacterium with antibiotic resistance, therebyidentifying the antibiotic resistance gene.
 34. The method of claim 30wherein the genome of said antibiotic resistant bacterium and the genomeof said wild-type bacterium are compared by: introducing gene fragmentsfrom said wild-type strain of said bacterium into the antibioticresistant strain of said bacterium; selecting a mutant bacterium withantibiotic resistance; and sequencing said gene fragment from saidmutant bacterium, thereby identifying the antibiotic resistance gene.35. A method of producing an antibiotic resistant bacterium comprisingthe steps of: culturing bacteria with a natural defect in mismatchrepair; contacting said bacteria with at least one antibiotic; selectinga bacterium among said bacteria resistant to said antibiotic; andculturing said bacterium; thereby generating antibiotic resistantbacteria.
 36. A method of generating antibiotic resistant bacteriacomprising the steps of: overexpressing a mismatch repair gene in abacterium whereby said bacterium becomes hypermutable; contacting saidbacterium with at least one antibiotic; determining whether saidbacterium is resistant to said antibiotic; and culturing said bacterium;thereby generating antibiotic resistant bacteria.
 37. The method ofclaim 36 further comprising making said antibiotic resistant bacteriagenetically stable.
 38. An antibiotic resistant bacterium produced bythe method of claim
 1. 39. An antibiotic resistant bacterium produced bythe method of claim
 35. 40. An antibiotic resistant bacterium producedby the method of claim
 36. 41. An antibiotic resistant bacteriumproduced by the method of claim 37.